# Patent

## Title
Heat-resistance rice gene OsZFP, screening marker and separation method thereof

## Abstract

Provided in the present invention are a separated endogenous rice gene resistant to high temperature (hereinafter referred to as the OsZFP gene for short) and a polypeptide encoded thereby, optimizing rice cells comprising the heat-resistant gene of the present invention or the polypeptide encoded thereby, and the plant cell preparation method thereof. Further provided are new methods and technologies for breeding new varieties of heat-resistant crops, comprising the related regulatory sequence for heat-resistance and a closely linked molecular marker denoting the heat-resistant gene and the sequence thereof.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part of International Patent Application No. PCT/CN2013/001504 filed Dec. 5, 2013 which claims priority to Chinese Application No. 201210515449.2 filed Dec. 5, 2012, the entire content of each of which is incorporated herein by reference in its entirety.


STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 9363-21TSIPv2_ST25.txt, 18,333 bytes in size, generated on Sep. 25, 2015 and filed via EFS-Web, is provided in lieu of a paper copy. The Sequence Listing is incorporated herein by reference into the specification for its disclosures.
TECHNICAL FIELD
The present invention relates to the field of plant genetic engineering and molecular breeding technology. More particularly, the present invention relates to mapping, isolation, functional analysis, sequence characterization of heat tolerance genes, identification of regulatory sequences, as well as screening and creation of co-segregating molecular markers, and their uses for improving the stress resistance of crops such as rice, and genetic transformation of a heat tolerance gene into rice and molecular marker-assisted method and procedure for selection. The invention further relates to use of the heat tolerance gene in assistance of improving the stress resistance of rice and other crops.
BACKGROUND ART
Rice is the most important food crop, and is the main source of food for more than half of the population all over the world (Khush, 2005). It is reported that an annual increase rate of 0.6 to 0.9% in rice yield is needed to meet the needs of people's lives (Carriger and Vallee, 2007). The increase of crop yield depends on farmland, and the sustainable development of agricultural production requires avoidance of environmental deterioration, destruction of ecological balance and loss of biodiversity (Cassman, 1999; Tilman et al., 2002). Crop yield is mainly determined by photosynthesis and respiration, however, both photosynthesis and respiration are sensitive to temperature (Yoshida, 1981), and also influenced by CO2 concentration in the atmosphere (Baker et al., 1990) and ozone layer (Maggs and Ashmore, 1998). These factors are also related to the greenhouse effect (Rosenzweig and Parry, 1994). Continual climate warming has brought devastating impacts on rice production. For two consecutive years 2006 and 2007, high temperature weather above 38° C. occurred for a long term in a large area along the middle and lower Yangtze River, including such as Chongqing, Hubei, Hunan, Anhui, Zhejiang, and Guangdong provinces, leading to large-scale drop of yield of rice and other crops in a large area, with ripening rate lower than 50% in the severe cases. Therefore, there is an urgent need for available heat tolerance genetic resources in breeding to respond to the serious threat of rice production caused by global warming. Therefore, it is of great significance in both theory and practice to carry out an extensive research on high temperature resistance, develop further heat tolerance genetic resource, and make efforts to select and breed new plant varieties with high temperature resistance, so as to respond to the serious threat of rice production caused by global warming.
In recent years, a large number of studies of genetic analysis and chromosomal mapping have been carried out at home and abroad regarding the high temperature resistance at the rice booting stage (Zhigang Zhao et al., 2006), heading stage (Yongsheng Zhang, et al., 2009; Zhang et al., 2009.), flowering stage (Liyong Zhao et al., 2003, 2002; Yi Pan et al., 2005; Qingquan Chen, 2008; Tao Zhang et al., 2008; Limei Que et al., 2008; Zhang et al., 2009; Jagadish et al., 2010.), filling stage (Changlan Zhu et al., 2005) and the stage for rice amylose synthesis and gel consistency formation (Changlan Zhu et al., 2006). In these studies, the mapped rice high temperature resistance related quantitative trait loci (referred to as QTL) relates to each chromosome of rice, wherein the QTL site with the most significant genetic effects is from variety Bala from Pakistan, which is responsible for 18% of phenotypic variation. However, since different researchers use different test materials and different high temperature treatment procedures and methods, the experimental data obtained are difficult to correspond to each other and reproduce, and the mapped QTL interval is relatively large, making it impossible to determine candidate genes and carry out the related molecular cloning and functional complementary verification. Therefore, the above mapped high temperature resistance related QTLs, not only theoretically lack support by the necessary data, but also are difficult to be used in practice effectively.
In addition, some researchers also carried out studies on rice high temperature resistance related genes by homologous cloning methods. Yamanouchi et al., (2002) used map-based cloning method for mapping, and cloned a rice spot gene Sp17. It is found that one of reading frame of the gene is highly similar with heat stress transcription factor. Under conditions of heat stress, the expression amounts of mutant and wild-type Sp17 are both up-regulated. Yokotani et al., (2008) transferred heat resistant gene encoding OsHsfA2e of rice into Arabidopsis thaliana and the tolerance of the transgenic A. thaliana to environmental stress is enhanced. According to the microarray analysis on transgenic A. thaliana plant with over-expression under non-stress condition exhibited increased expression amounts of some genes related to stress, including several types of heat shock proteins.
It is generally believed that heat shock proteins (HSPs) are associated with the high temperature response. It has been reported that rHsp90 responds to several stresses such as salt, drought and high temperature, and high temperature treatment at 42° C. and 50° C. for 30 minutes can significantly increase the rHsp90 expression amount (Liu et al., 2006). There is another report showing that there are 40 genes encoding proteins containing α-crystals, 23 of which are heat shock proteins (Sarkar et al., 2009). The microarray and RT-PCR analyses show that the expression amounts of 19 out of 23 heat shock proteins are up-regulated at high temperatures. In addition, Chang et al. (2007) transferred the rice heat shock protein Hsp101 into tobacco, and found that at high temperatures, the survival of the plant with over-expression is better than that of wild-type. Wu et al. (2009) drove OsWRKY11 expression with HSPIO promoter and found that the transgenic rice plants have relatively slower leaf wilting and a high survival rate after heat treatment. By over-expressing rice OsCEST (chloroplast protein capable of enhancing stress resistance) gene in A. thaliana, Yokotani et al. (2011) found that the transgenic plant is resistant to not only salt stress, but also is to drought and high temperature.
Zinc finger protein is a large family of transcription factors, which play important roles in gene expression regulation, cell differentiation, embryonic development and other biological processes (Gerisman & Pabo, 1997; Laity et al., 2001), especially in the expression regulation of stress related genes (Li & Chen, 2000). According to the number and location of cysteine (C) and histidine (H) residues in zinc finger protein, the transcription factors containing zinc-finger protein domains can be classified into subclasses C2H2, C2C2, C3H, C3HC4 (i.e., RING finger), C3HC5 (i.e., LM finger) and others. Among them, C2H2 zinc finger proteins are of the most clearly studied class among the zinc finger proteins, wherein two cysteines and two histidines form a coordinate bond with Zn2+, thereby forming in turn a tight finger structure containing one β fold and one α-helix. Kim et al. (2001) isolated a cold-inducible zinc finger protein gene SCOF-1 from the soybean cDNA library, which encodes a product containing two typical C2H2 zinc finger structures. The expression of SCOF-1 is specifically induced by cold and ABA, rather than salt stress. The transgenic study confirmed that the over-expression of SCOF-1 can enhance the cold resistance of A. thaliana and tobacco. Liu M. et al. (2007) cloned a soybean C2H2 zinc finger protein transcription factor gene GmC2H2, whose expression is related with the stress induction of cold and ABA. As for C3HC4 and CHY zinc finger proteins, the successful isolation are only reported in A. thaliana, rice, Physcomitrella patens, Artemisia desertorum, corn, pineapple, soybeans and other plants (Stone et al., 2005; Ohyanagi et al., 2006; Rensing et al., 2008; Yang et al., 2008; Alexandrov et al., 2009; Yang X. et al., 2009; Wu X. et al., 2010). A. desertorum AdZFP1 gene is a typical example that encodes such zinc finger proteins (Yang et al. 2008), and the semi-quantitative PCR analysis showed that, AdZFP1 gene is strongly induced by exogenous ABA, and to some extent is also induced by high salt, low temperature and high temperature. Wu X. et al. (2010) also screened a C3HC4 zinc finger protein gene GmRZFP1 from the cDNA library of the soybeans under drought condition, and the results demonstrated that the gene is mainly induced by high temperatures and drought stress. During the high temperature stress for 1-6 hours, the expression amount of GmRZFP1 Gene is positively correlated with treatment duration. In particular, under the high-temperature stress for 12 hours, the expression amount decreased, while the expression reached the highest level at 24 hours. These results therefore showed that GmRZFP1 gene is induced by a variety of stress treatments, probably involving in multiple stress signal transduction.
In addition, Huang et al. (2008) found 12 A20/AN1 type zinc finger proteins from the Japonica. The microarray analysis showed that the expressions of four genes (ZFP177, ZEP181, ZFP176, ZFP173), two genes (ZFP181 and ZFP176) and one gene (ZFP157) are induced by cold, drought and H2O2, respectively. Further study shows that ZFP177 responds to both low temperature and high temperature stress. By over-expressing ZFP177 gene, the obtained transgenic tobacco resists to low temperature of 2° C. and high temperature of 55° C., but becomes more sensitive to salt stress and drought stress, suggesting that ZFP177 plays an important role in various abiotic stresses in plant, while different stresses may have different response mechanisms.
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SUMMARY OF THE INVENTION
To solve the above problems, through the intensive studies, the present inventors have developed an optimized method for mapping the stress-resistance gene in plants, especially the high temperature-resistant gene, provided a number of new markers for genetic screening and mapping, and precisely mapped a new high temperature-resistant gene in rice, thereby providing a breeding method for converting a temperature-sensitive plant to a non-temperature-sensitive plant, and converting an ordinary plant to a plant with resistance to high temperature. In particular, the present invention relates to the following aspects:
1. A polypeptide, selected from any one of the following:
A) a polypeptide comprising an amino acid sequence as shown in SEQ ID NO:3;
B) a polypeptide comprising an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO:3 by deleting, substituting or inserting one or more amino acids; and having a function of providing high temperature resistance;
C) a polypeptide having an amino acid sequence as shown in SEQ ID NO:3; and
D) a polypeptide of an amino acid sequence as shown in SEQ ID NO:3.
2. A gene, wherein the polynucleotide sequence of the gene is a polynucleotide sequence selected from any one of the following:
A) a polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:1;
B) a polynucleotide comprising a polynucleotide sequence obtained by keeping the nucleotides corresponding to those at positions 2360 to 2371 of the nucleotide sequence as shown in SEQ ID NO:1 unchanged as GGGGGGGGGGGG (SEQ ID NO:12), and subjecting one or more nucleotides at the other positions of the nucleotide sequence to deletion, addition, or insertion; and encoding a polypeptide having a function of providing high temperature resistance;

C) a polynucleotide having a nucleotide sequence as shown in SEQ ID NO:1;

D) a polynucleotide of a nucleotide sequence as shown in SEQ ID NO:1; and

E) a polynucleotide sequence encoding any one of the polypeptide according to item 1.

3. A vector comprising the gene of item 2.

4. A host cell, characterized in that said cell comprises the polypeptide according to item 1, or the gene according to item 2, or the vector according to item 3, wherein the host cell is preferably an eukaryotic cell, more preferably a plant cell and a yeast cell, the plant cell is preferably a Gramineae plant cell, particularly preferably a rice (Oryza sativa L.) cell.

5. A molecular marker for screening, mapping and isolating new nucleotide sequences, wherein the marker is selected from:

A) insertion/deletion marker InDel5, located between 9130-9150 kb from the short arm terminus on chromosome 9 of rice, and the amplification product of the marker has a length polymorphism;

B) SNP marker, referred as RBsp1407, corresponding to the sequences of TGT705ACA and TGG705ACA in high temperature sensitive and high temperature resistant rice plants, respectively, based on the polynucleotide sequence of SEQ ID NO:1;

C) Microsatellite DNA marker RM7364, located between 9440-9450 kb from the short arm terminus on chromosome 9 of rice, and the amplification product via primers of the marker has a length polymorphism.

6. Use of the marker of item 5 for screening, mapping and isolating high temperature sensitive/resistive genes, wherein the high temperature is preferably 42° C. or higher, 45° C. or higher, 48° C. or higher, or 50° C. or higher.

7. A method of plant breeding, said method comprising applying the polypeptide according to item 1, or applying the gene according to item 2, or the vector according to item 3, or the host cell according to item 4, or the marker according to item 5 or 6.

8. A method of converting a temperature-sensitive rice to a non-temperature-sensitive rice, the method comprising mutating the nucleotide corresponding to the nucleotide at position 705 of SEQ ID NO:1 on chromosome 9 of the genome of the rice with the temperature-sensitive phenotype to G by genetic manipulation.

9. A method of inducing a high temperature resistant phenotype in rice, the method comprising inserting G at the 5′ end of the position corresponding to position 2360 of SEQ ID NO:1 on chromosome 9 of the genome of the rice without the temperature-resistant phenotype by genetic manipulation, wherein the high temperature is preferably 42° C. or higher, 45° C. or higher, 48° C. or higher, or 50° C. or higher.

10. A method of converting a temperature-sensitive rice to a high-temperature resistant rice, the method comprising mutating, by genetic manipulation, the nucleotide corresponding to the nucleotide at position 705 of SEQ ID NO:1 on chromosome 9 of the genome of the rice with the temperature-sensitive phenotype to G, and inserting one G at upstream from the 5′ end of the position 2360 in chromosome 9 of the genome of the temperature-sensitive rice, so as to obtain a nucleotide sequence being the same as the nucleotides from positions 2360 to 2371 in SEQ ID NO:1, i.e., the sequence comprises 12 continuous Gs from position 2360 in the direction away from the start codon, wherein the high temperature is preferably 42° C. or higher, 45° C. or higher, 48° C. or higher, or 50° C. or higher.

11. The present invention relates to a method of identification of high temperature resistance in plants, the method can be summarized as follows: subjecting a soil-cultivated seedling at two-leaf stage to a three-leaf stage to a high temperature treatment at 45-48° C. for 79 h, observing the results of the temperature responses after recovery for 5 days; as a standard procedure for the identification of high temperature resistance, the relative humidity and other cultivation conditions are set on the same level.

12. The method according to item 11, preferably further comprising using a pot for each cultivation with a uniform size (length 43 cm; width 33 cm; height 10 cm), weighing and adding the same amount of potting soil with a cultivation scale of 5×12 seedlings/each variety with one line of high temperature material positioned around as a protective line to eliminate the marginal effect, leaving a clear boundary between the protective line and the formal experimental materials; during the treatment, the humidity in the growth chamber is set at 75%, and length of light/dark cycle is set at 12 h.




BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 High temperature stress treatment assay and the response of the high temperature resistant and sensitive varieties HT54 and HT13 to the high temperature stress. Shown in FIG. 1 is growth status of the soil-cultivated seedlings at two-leaf stage, which has been subjected to different high temperature treatments for 79 hours and recovered for 5 days. Panel A: high temperature treatment at 42° C.; Panel B: high temperature treatment at 45° C.; Panel C: high temperature treatment at 48° C. It can be seen from the photographed results that the high temperature resistant variety HT54 and the high temperature sensitive variety HT13 exhibit a substantive difference in terms of survival and death after high temperature treatments at 45° C. and 48° C.
FIG. 2 The response of the high temperature resistant and sensitive varieties HT54 and HT13 to the high temperature stress. The treatment temperature is 48° C. The duration of the treatment is 84 hours. The treatment is performed at two-leaf stage. The seedlings are cultured in water. The figure shows the survival statues following a recovery period of five days after the treatments.
FIG. 3 The preliminary detection results of the linkage group of high temperature resistance locus in hybrid F2 generation of HT54 (high temperature resistant)×HT13 (high temperature sensitive). The molecular marker used is SSR marker RM444 located in the ninth linkage group of rice. In the figure, P1 represents high temperature resistant parent; P2 represents high temperature sensitive parent; R represents DNA pool of high temperature resistant extreme individuals; and S represents DNA pool of high temperature sensitive extreme individuals.
FIG. 4 The linkage genetic map of heat tolerance loci of HT54. (Panel a): the linkage genetic map generated by preliminary linkage group analysis on the DNA pool; the numbers in the figure represent the map distance between the markers; (Panel b) the genetic linkage map generated by densifying the molecular markers with a mapping population of 61 plants; the numbers in the figure represent the map distance between the markers; (Panel c): the genetic linkage map generated by further densifying the molecular markers with an expanded mapping population of 131 plants; the numbers in the figure represent the physical distance between the markers.
FIG. 5 The results of the detection of the exchanged plants (appearing in the mapping population during the mapping process using InDel5 and RM7364 markers) with PCR-RFLP marker exhibiting one single nucleotide polymorphism (SNP) change in the candidate genes (ZFP) sequence between the high temperature resistant and sensitive parents. In this figure, M, RP, SP, S represent molecular weight marker (DL2000), the high temperature resistant parent HT54, the high temperature sensitive HT13 and three exchanged plants.
FIG. 6 The dynamic expression pattern analysis of high temperature resistant and sensitive ZFP alleles in response to 45° C. high temperature stress treatment. The analytical method used is real-time quantitative PCR analysis with rice actin gene acts as an internal standard in a PCR amplification of 25 cycles. In the figure, the four rows from top to bottom are HT54 (resistant)/actin, HT13 (sensitive)/actin, HT54 (resistant)/ZFP, HT13 (sensitive)/ZFP, respectively. It can be seen from the figure that the expression levels of ZFP in the 6 h-treated high-temperature resistance and sensitive samples are up-regulated and down-regulated, respectively.
FIG. 7 The subcellular localization analysis of the sequence encoded by of the heat tolerance gene OsZFP. The figure shows that: compared with the control 35S-YFP, 35S-ZFP-YFP, i.e., a fusion protein of candidate gene and yellow fluorescent protein, is mainly localized on the cell membrane.
FIG. 8 The DNA sequence (SEQ ID NO:1), encoding sequence (SEQ ID NO:2) and the amino acid sequence (SEQ ID NO:3) of its expression product of the heat tolerance gene OsZFP according to the present invention.



DETAILED DESCRIPTION OF THE INVENTION
To solve the above problems, the inventors have made intensive studies and achieved the present invention on the basis of a large number of test results. The present invention will be described in detail in combination with the accompanying drawings.
I. Standardized Procedure for Identifying High Temperature Resistance of Rice at Seedling Stage
First, the inventors establish a standardized procedure of for identifying high temperature resistance of rice at seedling stage. The outstanding advantages of this identification procedure are clear typing of high temperature resistant and sensitive varieties, good reproducibility, and excellent applicability. Therefore, the establishment of the procedure will provide a good technical basis for screening, evaluating high temperature resistant rice germplasm material, genetic analysis of heat tolerance genes, and chromosomal mapping and cloning. See Example 1 for details of the identification.
II. Molecular Mapping of Rice Heat Tolerance Genes
Based on the above test results, the inventors use high temperature resistant rice line HT54, high temperature sensitive rice line HT13 and the progeny hybrids F1 and F2 thereof as exemplary test materials to perform molecular mapping of rice heat tolerance gene.
Test Method
(1). Screening of Polymorphism Markers in Parents
The molecular mapping of rice heat tolerance gene is performed with microsatellite DNA length polymorphism markers (SSLP) with high polymorphism and good reproducibility. The SSR primers used are based on the primer sequences published in the Gramene database and the NCBI database and synthesized by Shanghai Sangon Service Company.
Rice genome DNA extraction is performed according to the small amount of DNA extraction method reported by McCouch et al. (1988). Specifically, the method comprises the following steps: 1) A small piece of leaf with a length of 4-5 cm is sampled, added with 700 μL 1.5×CTAB (containing 1.5% CTAB, 75 mM Tris-HCl, 15 mM EDTA, 1.05 M NaCl), and thoroughly ground; 2) the homogenate is transferred into a 1.5 ml centrifuge tube, incubated in a water bath at 56° C. for 20 min and cooled to room temperature; 3) an equal volume of chloroform: isoamyl alcohol (24:1) is added and shaken till uniform; 4) the sample is centrifuged at maximum speed (13200 rpm) for 10 min; 5) the supernatant is transferred to a new centrifuge tube, and added with two volumes of ice-cold 100% ethanol, stands for 20 min followed by collection of DNA by centrifugation; 6) the supernatant is discarded and the DNA is air-dried, added with 50-100 μL double-distilled water, and detected in UV spectrophotometer. A series of DNA working solution with concentration ranging from 50-100 ng/μL is prepared by diluting the DNA based on the determined concentration, and stored at 4° C. refrigerator for further uses.
2) Construction of Mapping Populations
In autumn of 2008, a hybrid F1 was prepared with the high temperature resistant material HT54 as male parent and the high temperature sensitive material HT13 as female parent in the experimental base of the Institute of Rice, Guangdong Academy of Agricultural Sciences. The female parent HT13 is treated via “warm-both emasculation” approach. Considering that HT13 is a temperature sensitive material, the temperature of the both is set at 2° C. lower than the normal case in order to avoid damage to the pistil thereof. Specifically: in the heading stage of rice, at around 7:30 am in sunny morning, a panicle of the female parent HT13 (2⁄3 is taken out) is selected, placed into a water bottle preset at 43° C. for 8 min (the bottle is sealed with cotton after the placement), then taken out and shaken off water droplets. The spikelets (have been pollinated or will bloom in a few days) which are not blooming, should all be removed and the rest are immediately coated in a paper bag. At around 9:00 am, panicles of the male parent HT54 are taken and inserted in a bottle containing tap water, and covered with a black cloth. One hour later, most florets of the male parent are in full bloom. The pollen is transferred to the blooming stigma of the female parent which has experienced high-temperature emasculation. The pollination is repeated for 2-3 times. Once the pollination is completed, the stigma of the female parent is coated with a brown paper bag, which is then clamped with a clip, which is kept away from the stigma. The paper bag is marked with a card, which recites the name and hybridization date. About eight days later, the paper bag is removed and the F1 seeds are harvested when it is ripe.
In the winter of 2008, the hybrid F1 is planted in Hainan province. Once the plants grow up, the leaves are taken for DNA extraction. 3-4 pairs of SSR markers with polymorphism in the two parents are used to determine whether the plant is a true hybrid, and after the plant heading, F2 seeds harvested after self-hybridization of F1 are used for the subsequent identification of high temperature resistance. The genetic analysis of a heat tolerance gene is performed with resistance/sensitivity ratio after high temperature treatment and then the heat tolerance genes are mapped.
3) High Temperature Treatment Procedure
The high temperature treatment of the parents, F1 or F2 is performed following the above seedling cultivation method and standardized high temperature treatment procedure. The specific steps are: a. nursery pots preparation, that is, each nursery pot (size 43 cm×33 cm×10 cm) is added with equal amount of sieved and well mixed paddy soil; b. sowing, that is, the germinated seeds are spaced sowed at 11 rows×18 seeds/pot. Meanwhile, the parents are sowed at 1 row/pot as a control with 2 rows positioned around as protective rows. Fertilizer and water managements are same as those of the rice in pot; C. sampling, that is, once the seedling grows up to three-leaf stage, a piece with a length of about 2-3 cm from the second leaf per plant is placed −80° C. refrigerator for subsequent DNA extraction of the identified sensitive plants. D. After two days of recovery, i.e., one day before the complete growth of the third leaf, the seedling is transferred into the artificial climate chamber for high temperature treatment. The artificial climate chamber is set as follows: 33° C. 1 h (Light, abbreviated as L, the same below)→36° C. 1 h (L)→39° C. 1 h (L)→42° C. 1 h (L)→45° C. 1 h (L)→48° C. 7 h (L)→48° C. 12 h (Dark, abbreviated as D, the same below)→48° C. 12 h (L)→48° C. 12 h (D)→48° C. 12 h (L)→48° C. 12 h (D)→48° C. 12 h (L). Humidity is set at 75%.
(4). PCR Program and Detection
SSR analysis is mainly revised by reference to the method of Wu et al. (1993) with a PCR system of 25 μL: 2.5 μL 10× buffer (Mg2+); 0.5 μl 10 mM dNTP; 1.0 μl 5 μM 5′-primer; 1.0 μl 5 μM 3′-primer; 0.5 μl Taq polymerase; 1.0 μl DNA; 18.5 μl ddH2O. dNTP is purchased from Shanghai Sangon Company, and Taq polymerase is purchased from Dingguo Company. PCR amplification program is: denaturation 94° C. for 5 min, then 35 cycles of (denaturation at 94° C. for 45 s, annealing at a temperature of 50-60° C. suitable for the selected primers for 45 s, extension at 72° C. for 45 s), followed by extension at 72° C. for 10 min. The amplification product is detected with 3% agarose gel electrophoresis (AGE) or 4% denaturing polyacrylamide gel electrophoresis (PAGE).
(5) Preliminary Mapping of the Heat Tolerance Gene
F2 segregating population is selected to establish a mapping population for gene mapping using a recessive extreme population method with microsatellite DNA polymorphism molecular marker (also called simple sequence length polymorphism, SSLP) as molecular marker. The specific procedure is as follows: the F2 segregating population is subjected to high temperature treatment, and the selected individual plant exhibiting high temperature sensitivity is used as a mapping population for gene mapping. Then, from the F2 segregating population, 10 high temperature resistant or sensitive plants are randomly selected. Their leaves are mixed in equal amount and grinded for DNA extraction to establish heat tolerance and sensitivity DNA pools. Thereafter, the two DNA pools are subjected to marker analysis using SSLP markers with polymorphism between the parents (Chen et al., 1997; Temnykh et al., 2000) to screen out the marker with polymorphism between the two DNA pools, whereby, the linkage group of the gene is preliminarily identified. On this basis, the mapping population established on the recessive extreme individuals in the above F2 segregating generation is subjected to genotype analysis individually using the polymorphism marker obtained in the DNA pools, and the obtained data is used for preliminary mapping of target genes.
(6) Fine Mapping of Heat Tolerance Gene
On the basis of preliminary mapping, the mapped segment is further analyzed with concentrated markers, until such markers are exhausted on the linkage group related to target gene. The SSLP markers used is either obtained from Gramineae genome database, or designed and synthesized in house according to the genomic sequence. Then, the biological function prediction and molecular characterization (including motifs, functional domains, conserved sequences and control sequences, etc.) analysis are performed on the coding sequence within the marker interval (usually at least less than or equal to 0.5 Mb) with biological information, in order to determine the candidate genes. Then, specific primers are designed for the candidate genes, and the candidate genes are cloned by TA cloning and sequenced. Finally, the target gene is predicted depending on the presence or absence of the difference.
(7) Calculation of Genetic Distance
The results of the electrophoresis images are converted with numerical statistics: those consistent with HT54 banding pattern are marked as A, those consistent with HT13 banding pattern are marked as B, those with the banding pattern of both parents are marked as H, and those with no banding are marked as -.
Genetic linkage analysis is performed using MAPMAKER3.0 software (Lander et al., 1987) for analysis and calculation of genetic distance (cM), genetic mapping with Mapdraw2.1 (Renhu Liu and Jinling Meng, 2003) and mapping analysis of heat tolerance genes.
For precise mapping of the genes, the inventors have developed a new mapping marker. The exemplary preparation of the marker is shown in Example 2.
The results of genetic analysis and gene mapping
1) Screening of Parental Polymorphism and Establishment of Molecular Map
The parents are screened for polymorphism using a total of 2304 SSLP molecular markers, from which 322 SSLP molecular markers with polymorphism between the parents are selected. The distribution of these markers on chromosome is shown in FIG. 6 in the scale of physical distance. The obtained polymorphism SSLP markers are relatively uniformly distributed in each chromosome, besides chromosome 9 and the region near the centromere.
The microsatellite molecular markers (SSLP) genetic linkage map established by rice heat tolerance gene mapping is shown in FIG. 2. The distance between the markers is physical distance.
2) Genetic Analysis Confirmed that the High Temperature Resistance of HT54 Seedling is Controlled by Major Dominant Single Gene
The parents, F1 and F2 hybrids at two-leaf stage are subjected to high temperature treatment using standardized procedure. It is shown that: F1 plants completely survive high temperature treatment, and F2 population exhibits significant segregation into high temperature resistance and sensitivity, in which there are 442 high temperature resistant plants and 152 high temperature sensitive plants, consisting with the segregation ratio of 3:1 (see Table 1, Chi-square test, P>0.05, indicating that the difference is not significant). The results thus demonstrated that the high temperature resistance of HT54 at seedling stage is controlled by a major dominant single gene. The gene is now named as OsHTAS (Oryza sativa Heat Tolerance at Seedling stage).







TABLE 1







Genetic analysis of the high temperature resistance of HT54











No. of high
No. of high





temperature
temperature
Total No.
X2 (3:1)


resistant plants
sensitive plants
of plants
test
P value





442
152
594
0.11
0.70-0.80









3) Preliminary Mapping Shows that the Heat Tolerance Gene is Located in the Ninth Linkage Group of Rice

Among 322 pairs of SSLP markers with polymorphism between the parents, only RM444 exhibits heterozygous banding pattern during the detection of heat tolerance DNA pools, and the heat sensitivity DNA pool is consistent with the high temperature sensitive parent, suggesting that RM444 may be linked with the target gene (see FIG. 3). In addition, in the PCR amplification products of F1, the intensity of band amplification is basically consistent between the two parents. The banding pattern of the heat tolerance DNA pool is heterologous, but the banding pattern of HT54 is obviously stronger than that of HT13, suggesting that the heat tolerance DNA pool may contain both high temperature resistant homozygote and heterozygote. The banding pattern of the heat sensitivity DNA pool is essentially HT13 banding pattern, indicating that the DNA pool is mainly composed of the DNA of high temperature sensitive recessive homozygous individual plants, thus suggesting that the molecular marker used may be linked with target heat tolerance gene. Then, 61 recessive extreme high temperature sensitive individuals and 40 high temperature resistant individuals are selected from F2 high temperature-treated population to establish two validation populations for RM444 marker analysis. The results show that: among 61 recessive extreme high temperature sensitive individuals, 13 plants have heterozygous banding pattern, 43 plants have HT13 banding pattern, 5 plants have no banding pattern, and no plant has HT54 banding pattern; and among 40 high temperature resistant individual plants, 23 plants have heterozygous banding pattern, 2 plants have HT13 banding pattern, and 15 plants have HT54 banding pattern. These results thus further confirm that the RM444 marker is linked with heat tolerance gene OsHTAS.
4) Fine Mapping Further Demonstrates that Heat Tolerance Gene is Located Between the Two Markers RM7364 and InDel5 and the Actual Physical Distance of the Interval is 420 bp.
Primers on the short arm terminus (i.e., upstream of RM444) of chromosome 9 are retrieved from the Gramene database for parent marker typing analysis. Three SSLP markers with polymorphism between the parents are identified, i.e., RM23687, RM23719 and RGNMS2991. Upon scanning of the previously-used mapping populations of 61 individuals with these three markers, it shows that RM23719 and RM23687 are indeed linked with the heat tolerance gene OsHTAS (FIG. 4a). Their linkage distances are 12.2 cM and 13.1 cM respectively. No valid data for RGNM2991 is obtained. In addition, it is found that RM23719 is immediately located downstream of RM444, and the genetic distance therebetween is 0.9 cM (FIG. 4a), but the actual physical distance therebetween is 4.48 Mb. RM23687 is close to endpoints (the distance therebetween is 1.07 Mp), and the distance from RM23719 is 1.01 Mb, but the detected linkage genetic distance from heat tolerance gene OsHTAS is up to 17.9 cM. According to information of rice chromosome 9 listed in the database of Gramineae genome, it is found that the above abnormal linkage inheritance may be due to the reason that RM23719 is near the centromere. Thus, it is presumed that the heat tolerance gene may be downstream of RM444, i.e., the long arm terminus of rice chromosome 9.
Then, 114 pairs of SSLP primers are found by retrieving primers for the long arm terminus (i.e., upstream of RM444) of chromosome 9 in the database of Gramineae genome. Upon parent polymorphism analysis, 10 pairs of primers with polymorphism between the parents are identified, i.e., RM23982, RM23985, RM7364, RM24019, RM5777, RM240712, RM24075, RM24099, RM24102, and RM24170. The marker analysis on the heat tolerance and sensitivity DNA pools demonstrate that RM23982, RM23985, RM7364 and RM24019 also exhibit polymorphism between the heat tolerance and sensitivity DNA pools. These markers are used for further marker analysis on the mapping populations of 61 F2 individuals, which localizes the heat tolerance gene OsHTAS between RM23985 and RM7364 (see FIG. 8b). Without further SSLP marker known to be located in this region, 90 pairs of insertion/deletion (InDel) markers are designed according to the Indica/Japonica rice genome sequence information. Upon marker analysis of the parents, 8 pairs of primers with parent polymorphism are identified. However, only InDel3 and InDel5 have a large polymorphism between the parents and are convenient for the discrimination of the banding pattern of the mapping population.
The linkage genetic map of the heat tolerance gene OsHTAS on chromosome 9 is shown in FIG. 4.
The primer sequences for the two insertion/deletion markers are respectively as follows: InDel3F: 5′-GTTTGCGACATTGGAGCCTTC-3′ (SEQ ID NO:4) and InDel3R: 5′-AATGCTTGGGTATGCTAGGTGAA-3′ (SEQ ID NO:5); InDel5F: 5′-TCCTCGGAGATGTTTGACCTTG-3′ (SEQ ID NO:6) and InDel5R: 5′-CAGAAGGTG TACGCAACTCTTGT-3′ (SEQ ID NO:7).
Thus, the rice heat tolerance gene OsHTAS is further mapped between RM7364 and InDel5 using these two in-house designed indel markers. The genetic distance of the gene and these two closely linked markers are 2.5 cM and 1.7 cM, respectively, and the actual physical distance between the markers is 420 Kb. Then, the mapping populations are expanded to 131 individuals, and the determined genetic linkage distance between heat tolerance gene OsHTAS and InDel3, InDel5, RM7364, RM24019 is 4.0 cM, 3.2 cM, 1.2 cM and 1.6 cM, respectively (FIG. 4b). These mapping results are essentially consistent with those of the mapping population of 61 individual plants as described above.
5) The Confirmed Candidate Gene is a Zinc Finger Protein Gene, which has Two SNP Differences Between the High Temperature Resistant and Sensitive Parents and is Co-Segregated with a PCR-RFLP Marker Developed Based on One of the SNP Differences.
Since it is difficult to achieve more useful polymorphism markers in the above mapping region, the existing biological database information in combination with the published genetic data are used to screen and identify candidate genes. A total of 60 known and unknown genes are retrieved by searching 420 Kb length DNA sequence of the mapping segments (from Indel5 to RM7364) in the website http://rice.plantbiology.msu.edu/, among which, 15 genes encode retrotransposable transposon proteins, 6 genes encode transposon proteins, 27 genes encode proteins of known functions, 12 genes encode proteins of unknown functions. It has been reported that: the relevant ubiquitin binding enzyme protein (LOC_Os09g15320) and zinc finger protein (LOC_Os09g15430) genes are associated with the high temperature resistance of rice. Therefore, they are preliminarily identified as candidate genes. Then, upon the analysis of the standardized microarray data, among the genes with expression value above 100 [nucleobase-ascorbate transporter (LOC_Os09g15170), retrotransposon protein (LOC_Os09g15250), ubiquitin-binding protein (LOC_Os09g15320), transporter family protein (LOC_Os09g15330), hydrolase (LOC_Os09g15340), NAD-dependent epimerase/dehydratase (LOC_Os09g15420), zinc finger protein (LOC_Os09g15430) and serine/threonine rich protein T10 in DGCR region (LOC_Os09g15480)], the ubiquitin binding protein (LOC_Os09g1532) and the zinc finger protein (LOC_Os09g15430) are both over-expressed, indicating the important role of these two genes in rice seedling growth and development. Thus, the cDNA sequence and the genomic DNA sequence of the promoter and terminator of these two candidate genes are amplified from the seedling leaf tissue of the high temperature resistant and sensitive parents and sequenced. As a result, it is found that there is no difference between the cDNA sequences of the high temperature resistant and sensitive parents, but there are single nucleotide polymorphism (SNP) differences localized, respectively, in the 5′-untranslated region and promoter sequence in the zinc finger protein (LOC_Os09g15430). Since the SNP difference in the promoter sequence leads to one change of the recognition site of the restriction endonuclease Bsp1407 (T ↓ GTACA), one PCR-RFLP (CAPs) marker is designed, referred to as RBsp1407.
The size of the labeled PCR amplification product is 580 bp. The amplified PCR product is recovered, and subjected to enzymatic digestion using endonuclease Bsp14071 enzyme (Promega). As a result, it is shown that: the amplification product of HT54 is not digested by the enzyme and the fragment is still 580 bp in size, while the amplification products of HT13 and all exchanged plants presented in the mapping population of 131 plants are digested by the enzyme, resulting two fragments in size of 422 bp and 158 bp, respectively (FIG. 5). The result that PRBsp14071 marker presents no exchanged plant between InDel5 and RM7364 demonstrates that: with the sizes of the mapping population used, the marker and the heat tolerance gene OsHTAS is co-segregated, thereby further identifying the candidate gene as zinc finger protein gene.
The technical solutions of the present invention will be described in detail by means of the following specific examples. It is obvious to those skilled in the art that the specific embodiments are provided to facilitate those skilled in the art to reproduce the exemplary technical information, and is not intended to limit the subject matters claimed in this application. It shall be understood that any modification to the exemplary technical solutions without departing from the spirit of the present invention is intended to be covered within the scope of the present invention, as long as the beneficial technical effect can be achieved.
EXAMPLES
Example 1: Identification of Heat Tolerance Genes
As exemplary test materials, a high temperature resistant rice variety HT54 and a high temperature sensitive rice variety HT13 (both of them are O. sativa ssp. indica) are employed in this example.
1) Cultivation of Rice Seedlings and Setting of High Temperature Treatment Conditions
(1). Cultivation of Seedlings
The soils are taken from paddy fields at the same area, air-dried naturally, crushed and sieved, distributed into plant growth pots (26 cm×18 cm×6 cm) with equal weight, and added an equal amount of water to soak overnight for seeding. The test materials are soaked, germinated and then seeded. Each pot is divided into two parts, one half for HT54 and the other for HT13, with 3 rows (8 seedlings/row) for each variety.
(2) High-Temperature Treatment Period and Treatment Temperatures as Well as Other Condition Settings
The high temperature treatment is carried out during the seedling stage. The artificial climate chamber used is the Intelligent Artificial Climate Chamber PRX-1000B produced by Zhejiang Ningbo Fu Experimental Instrument Factory. The temperatures are set at 42° C., 45° C. and 48° C. for three individual treatments, each increasing from 33° C. at a rate of 3° C./h until it reaches the set temperature. The humidity is set at 75% and the other cultivation conditions are set at the same level.
(3) Procedure for Identification of High-Temperature Resistant Rice and Setting of Standardized Parameters
The seedlings of the high temperature resistant rice variety HT54 and the high temperature sensitive rice variety HT13 cultivated in soil to two-leaf and three-leaf stages are transferred into growth chambers, then subjected to high temperature treatment at the 3 temperatures for 79 h, allowed to recovery for five days after the treatment. The responses of the seedlings to the high temperatures are observed. The results are shown in FIG. 1. It is clear from the results in FIG. 1 that: at the temperature of 42° C., both the high temperature resistant seedling HT54 and the high temperature sensitive seedling HT13 completely survive the treatment; and at the treatment temperatures of 45° C. or 48° C., the high temperature resistant variety HT54 and the high temperature sensitive variety HT13 exhibit a substantive difference in terms of survival and death after high temperature treatment (FIG. 1). However, it is apparent that 45° C. is not the maximum temperature to which the variety HT54 can resistant. Thus, 48° C. is finally selected as the temperature set for the standard procedure. Therefore, the procedure for identification of high-temperature resistant rice determined by this trail can be summarized as: the seedling cultivated in soil to two-leaf stage is subjected to a treatment under the condition of heat temperature at 48° C. and 75% relative humidity for 79 h, allowed to recovery for five days and the response of the seedling to high temperature is observed. Survival or not can serve as an index of high temperature resistance or sensitivity of rice. It can be seen from the results in the photographs that the responses of the high temperature resistant variety HT54 and the high temperature sensitive variety HT13 to the high stress of high temperature treatment at 45° C. and 48° C. exhibit a substantive difference in terms of survival and death.
Example 2: Confirmation of New Genetic Markers
The existing SSLP technology is apparently insufficient to achieve the requirement of precise mapping of heat tolerance genes in rice. To this end, the inventors have developed new mapping markers. In particular, the inventors search Insertion/Deletion (InDel) polymorphism sites by differential sequences between the whole genomes of japonica rice (Oryza sativa L. subsp. Japponic) Nipponbare and indica (Oryza sativa L. subsp. Indica) 9311 published by NCBI. According to the DNA sequences flanked by these sites, the suitable primers are designed using Primer5.0 and NCBI online Primer blast is used to guarantee the specificity of the primers.
TA Cloning and Sequencing Analysis
a) A-addition: Since the purified PCR product is obtained by amplification with enzyme Proybest, its ends are blunt and A-addition is needed in order to perform TA cloning. For the A-addition reaction system of 20 μL in total, the components and the volume thereof to be added are: 10×PCR buffer, 2 μL; Taq enzyme, 0.5 μL; dNTPs (10 mM), 0.5 μL; the purified PCR product 17 μL. Upon blending, the mixture is placed into a PCR instrument at 72° C. for 30 min, and then stored at 4° C. The A-addition product is purified using Axy Prep PCR Clean-up Kit (Axygen).

b) Ligation: The ligation is performed following the manufacture's instruction of pMD18-T Vector System kit (TaKaRa). 4 μL purified A-addition product is mixed with 5 μL ligation mixture (solution I) and 1 μL pMD18-T vector, and ligated at a constant temperature of 16° C. for 1-2 hours (conducted in a thermostatic machine).

c) Heat shock transformation of the ligation product into E. coli: the competent cells are taken from −80° C., added into 10 μL ligation mixture, gently shaken, and incubated on ice for 30 min; heat shocked in a water bath at 42° C. for 90 s, and immediately placed on ice for 2 min; thereafter 800 μL LB medium is added to each tube, cultured in a shaking table at 37° C. with low-speed shaking for 45 min (for bacteria recovery, the speed does not exceed 190 rpm); the bacteria are enriched and plated on an LB plate (Amp/IPTG/X-Gal) for screening; upon the complete absorption of the bacterial broth into the medium, the plate is turned upside down and incubated inverted at 37° C. overnight.

d) Identification of positive transformant clones: White colonies are picked and identified by PCR amplification with universal sequencing primers for the vectors. The PCR reaction system is same as described above, and the reaction procedure is: pre-denaturation at 94° C. for 5 min, then total 30 cycles of 95° C. for 30 s, 55C for 30 s, and 72° C. (the specific duration may vary according to the size of the target gene) for extension, followed by extension at 72° C. for 5 min.

e) Identification of PCR product by electrophoresis: the length of the PCR product of negative colony is 156 bp (only the vector sequence is amplified), while the length of the PCR product of positive colony is larger than 156 bp (the length of the target gene fragment+156 bp).

f) Sequencing of the positive clones: 5 mL LB broth containing 50 μg/mL Amp is added into a 50 mL centrifuge tube, inoculated with PCR positive colony, and cultured at 37° C. with 220 rpm shaking overnight; the cultured broth is transferred into a 1.5 mL centrifuge tube, and centrifuged at 12000 rpm for 2 min, the supernatant is discarded, and the centrifuge tube is placed upside down on absorbent paper to make the bacterial pellet as dry as possible; plasmid DNA is extracted using a plasmid extraction kit from Axygen. The extracted plasmid DNA is stored at −20° C. for further use. Meanwhile 10 μL bacteria broths corresponding to the plasmids are taken for verification via sequencing (Shanghai Invitrogen). For each gene, 3 positive transformants are verified by sequencing. The new marker of the present invention is derived from one SNP occurring in the promoter of a candidate gene in high temperature resistant and sensitive parents. In particular, the presence of the SNP leads to the change of the recognition site of a restriction endonuclease Bsp1407 I. The sequence thereof in the high temperature resistant parent is 5′-TGGACA-3′, and can not be recognized by Bsp1407 I, while the sequence thereof in the high temperature sensitive parent is 5′-TGTACA-3′, and can be recognized by Bsp1407 I. Therefore, the specific primers are designed targeting the SNP region for amplifying the corresponding genomic fragment, which is then digested with Bsp1407 I, thereby enabling the detection of the polymorphisms difference.

According to this principle, the sequences of the specific primers designed in this example are: BspF: 5′-CCATCCAAACACGCCCTAA-3′ (SEQ ID NO:8) and BspR: 5′-ATTGCCCCTTGCTATGG T-3′ (SEQ ID NO:9). The size of the amplified PCR product is 580 bp, and the two fragments resulting from the Bsp1407 I digestion of the PCR product of the high temperature sensitive parent are 422 bp and 158 bp in size, respectively. The PCR-RFLP marker thus developed is referred as RBsp1407.
During the mapping of the high-temperature resistant gene, the marker has been proved to be co-segregated with the target gene.
In the experiment for validating RBsp1407 marker, the marker is used by the inventors for carrying out a confirmatory test on three single recombinant plants, which arise from the analysis of a recessive extreme location population for the two tightly linked markers RM7364 and InDel5. As a result, the result identical to that of the recessive high temperature sensitive parent is achieved, as shown in FIG. 5. The inventors thereby have further developed insertion/deletion marker InDel5, which is located between 9130-9150 kb from the short arm terminus on chromosome 9 of rice, and has a length polymorphism between the high temperature resistant and sensitive parents; and microsatellite DNA marker RM7364, which is located between 9440-9450 kb from the short arm terminus on chromosome 9 of rice. For marker RM7364, primers are designed as: RM7364F: 5′-TTCGTGGATGGAGGGAGTAC-3′ (SEQ ID NO:10); and RM7364R: 5′-RGCGTTTGTAGGAGTGCCAC-3′ (SEQ ID NO:11). Similarly, it is found that the amplified product of the marker via the primers has a length polymorphism between the high temperature resistant and sensitive parents.
Example 3: Confirmation of Heat Tolerance Gene OsHTAS
Through the aforementioned gene mapping studies, the present inventors have found a new gene located on chromosome 9 of the rice genome, the dominant heat temperature resistant gene OsHTAS. The difference between the dominant heat temperature resistant gene OsHTAS and the recessive allele Oszfp (Oshtas) regarding their DNA sequences only lies in that of a single nucleotide polymorphism (SNP), which occurs in a motif associated with high temperature resistance, salt resistance and drought resistance in OsHTAS 5′-terminal un-translated region (11T12G), wherein there is an additional G in OsHTAS (TTTTTTTTTTTGGGGGGGGGGGG SEQ ID NO:13) compared with Oshtas (TTTTTTTTTTTGGGGGGGGGGG SEQ ID NO:14). The full length of the dominant heat tolerance gene (LOC_Os09g15430) OsHTAS genomic sequence is 4784 bp, and the full length of the coding sequence (CDS) thereof is 1245 bp, encoding a product of 414 aa, which belongs to the family of zinc finger proteins; the particular sequence of rice (Oryza sativa) ZFP gene is shown as SEQ ID NO:1 in the sequence listing.
During the particular trials, the inventors perform insertion, deletion, substitution at partial sites of the gene, and verify the functions of the resulting mutants. As a result, it demonstrates that the mutants resulting from the manipulations such as degeneracy of the genetic codon and mutation of partial sites maintain the same high temperature resistant property as in the case of the gene shown in SEQ ID NO:1. More particularly, a polypeptide encoded by a nucleotide sequence in which the nucleotides at positions 2360 to 2371 corresponding to those shown in SEQ ID NO:1 remain unchanged as GGGGGGGGGGGG (SEQ ID NO:12), while one or more nucleotides at the other positions of the nucleotide sequence are subjected to deletion, addition, or insertion, still has a high temperature resistant property. The insertion, deletion, and substitution are performed in accordance with the genetic manipulation methods known in the art, and the number of the changed nucleotides is preferably 1-100 nucleotides, more preferably 1-50, 1-20 nucleotides, more preferably 1-10 nucleotides. In addition, the inventors construct an expression vector by ligation of heat tolerance gene with an inducible promoter, which is transferred into host cells such as yeast DY1455, and Arabidopsis thaliana Columbia, thereby successfully obtaining positive transformants.
Example 4: Characterization of the Response of Heat Tolerance Gene OsHTAS to High Temperature Stress
Various high temperature resistant and sensitive varieties are persistently treated with high temperature of 45° C. for 12 hours, and at 12 time points (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 84 h, 96 h), samples are taken for RNA extraction. The expression analysis of the candidate gene OsHTAS is carried out by the real-time quantitative PCR. The results reveal that the dynamic expression mode of the candidate gene in the high temperature resistant variety HT54 is obviously different from that in the high temperature sensitive variety HT13. In HT54 the expression level of the candidate gene is up-regulated, while that in HT13 is down-regulated. The results as shown in FIG. 6 indicate that the candidate gene actively responses to high temperature stress. It can be seen from FIG. 6 that the expression levels of OsHTAS in the 6 h-treated high-temperature resistance and sensitive samples are up-regulated and down-regulated, respectively.
Example 5: Functional Verification of the Candidate Genes: Over-Expression
The full-length cDNA of OsHTAS was inserted into the overexpressing vector derived by rice ACTIN I promoter, which was introduced into the genome of Nipponbare using Agrobacterium-mediated method, obtaining five positive independent transformants. Then, using the aforementioned mentioned artificial climate chamber, the soil-cultivated seedlings of these positive independent transformants at the two-leaf to three-leaf stages are subjected to high temperature treatment at 48° C. for 79 hours, with wild-type plant as a control. Upon the completion of the treatment, the seedlings are removed out of the growth chamber for recovery under normal temperature conditions for 5 days, and then the response of the seedlings to high temperature treatment is observed and recorded. The results demonstrate that: upon the high temperature treatment, the over-expressed plants have an enhanced high temperature resistance and higher survival rate compared with the wild-type, further indicating that the selected candidate gene is indeed associated with high temperature.
Example 6: Functional Verification of the Candidate Genes: RNAi Knock-Out
A specific sequence of the CDS region and UTR region of candidate gene is inserted into the RNA interference vector pTCK303 in forward and reverse directions, introduced into the genome of the Nipponbare using Agrobacterium-mediated method, obtaining five positive independent transformants. Then, using the aforementioned artificial climate chamber, the soil-cultivated seedlings of these positive independent transformants at the two-leaf to three-leaf stages are subjected to high temperature treatment at 48° C. for 79 hours, with wild-type plant as a control. Upon the completion of the treatment, the seedlings are removed out of the growth chamber for recovery under normal temperature conditions for 5 days, and then the response of the seedlings to high temperature treatment is observed and recorded. The results demonstrate that: upon the high temperature treatment, the interfered plant has a reduced high temperature resistance and lower survival rate compared with the wild-type, further indicating that the selected candidate gene is indeed associated with high temperature resistance.
Example 7: Subcellular Localization Analysis of Candidate Gene OsHTAS
By gene gun bombardment of onion epidermis, the subcellular localization results under confocal microscopy show that compared with the control 35S-YFP, 35S-ZFP-YFP, i.e., a fusion protein of candidate gene and yellow fluorescent protein, is mainly localized on the cell membrane, as shown in FIG. 7. The results indicate that the product encoded by the candidate gene ZFP is a membrane protein, which is consistent with the conclusion that a considerable number of membrane proteins are involved in signal transduction of stress, indicating a relationship between the subcellular localization and the functional expression. The result shown in FIG. 7 demonstrates that: compared with the control 35S-YFP, 35S-ZFP-YFP, i.e., a fusion protein of candidate gene and yellow fluorescent protein, is mainly localized on the cell membrane.
In summary, the results of the application disclosed herein can provide those skilled in the art with further understanding of the basic genetic rules of the traits of high temperature resistance. Meanwhile, the mapping of the genetic loci of the high temperature resistance and identification and cloning of the candidate gene as well as the development of the co-segregated markers thereof would establish an excellent theoretical and material basis for the functional analysis of the subsequent candidate genes and the effective use in the molecular breeding.
Free Text of the Sequence Listing
SEQ ID NO. 1 OsHTAS genome sequence;
SEQ ID NO. 2 coding sequence of OsHTAS gene;
SEQ ID NO. 3 polypeptide sequence of OsHTAS protein;
SEQ ID NO. 4 artificial primer sequence;
SEQ ID NO. 5 artificial primer sequence;
SEQ ID NO. 6 artificial primer sequence;
SEQ ID NO. 7 artificial primer sequence;
SEQ ID NO. 8 artificial primer sequence;
SEQ ID NO. 9 artificial primer sequence;
SEQ ID NO. 10 artificial primer sequence;
SEQ ID NO. 11 artificial primer sequence;
SEQ ID NO. 12 sequence of nucleotides 2360 to 2371 from SEQ ID NO:1;
SEQ ID NO. 13 OsHTAS 5′-terminal un-translated region sequence; and
SEQ ID NO. 14 Oshtas 5′-terminal un-translated region sequence.

## Claims

1. A polynucleotide comprising the polynucleotide sequence obtained by keeping the nucleotides corresponding to those at positions 2360 to 2371 of the nucleotide sequence as shown in SEQ ID NO. 1 unchanged as GGGGGGGGGGGGG, and subjecting one or more nucleotides at the other positions of the nucleotide sequence as shown in SEQ ID NO. 1 to deletion, substitution, or insertion; and the number of changed nucleotides is 1-100 nucleotides, 1-50 nucleotides, 1-20 nucleotides, or 1-10 nucleotides; and encoding a polypeptide providing high temperature resistance to a host cell comprising the polypeptide; wherein the high temperature is 42° C. or higher, 45° C. or higher, 48° C. or higher, or 50° C. or higher.

2. A vector comprising the polynucleotide of claim 1.

3. A method for screening, mapping and/or isolating high temperature sensitive/resistive genes, comprising using
a molecular marker selected from:
A) insertion/deletion marker InDel5, located between 9130-9150 kb from the short arm terminus on chromosome 9 of rice, and the amplification product of the marker has a length polymorphism;
B) SNP marker, referred as RBsp1407, corresponding to the sequences of TGT705ACA and TGG705ACA in high temperature sensitive and high temperature resistant rice plants, respectively, based on the polynucleotide sequence of SEQ ID NO: 1;
C) Microsatellite DNA marker RM7364, located between 9440-9450 kb from the short arm terminus on chromosome 9 of rice, and the amplification product via primers of the marker has a length polymorphism;
thereby screening, mapping and/or isolating high temperature sensitive/resistive genes.

4. A method of converting a temperature-sensitive rice to a non-temperature-sensitive rice, the method comprising inserting the polynucleotide of SEQ ID NO:2 into an overexpressing vector driven by rice ACTIN I promoter, and introducing the vector into the genome of the temperature-sensitive rice using Agrobacterium-mediated transformation:
thereby converting the temperature-sensitive rice to a non-temperature-sensitive rice.

5. A method of inducing a high temperature resistant phenotype in rice, the method comprising inserting the polynucleotide of SEQ ID NO:2 into an overexpressing vector derived by rice ACTIN I promoter, and introducing the vector into the genome of the rice using Agrobacterium-mediated transformation, wherein the high temperature is 42° C. or higher, 45° C. or higher, 48° C. or higher, or 50° C. or higher;
thereby inducing a high temperature resistant phenotype in the rice.

6. A method of converting a temperature-sensitive rice to a high-temperature resistant rice, the method comprising inserting the polynucleotide of SEQ ID NO: 2 into an overexpressing vector driven by rice ACTIN I promoter, and introducing the vector into the genome of the temperature-sensitive rice using Agrobacterium-mediated transformation, wherein the high temperature is 42° C. or higher, 45° C. or higher, 48° C. or higher, or 50° C. or higher;
thereby converting the temperature-sensitive rice to a high-temperature resistant rice.

7. A host cell comprising the polynucleotide according to claim 1.

8. The host cell of claim 7, wherein the host cell is an eukaryotic cell.

9. The host cell of claim 7, wherein the host cell is a plant cell or a yeast cell.

10. The host cell of claim 7, wherein the host cell is a rice (Oryza sativa L.) cell.

11. A method of plant breeding, said method comprising inserting the polynucleotide according to claim 1 into an overexpressing vector driven by rice ACTIN I promoter, introducing the vector into the genome of the plant using Agrobacterium-mediated transformation, subjecting positive independent transformants to high temperature treatment at 48° C. for 79 hours, and selecting surviving plants having enhanced high temperature resistance.

12. A method of plant breeding, said method comprising culturing applying the host cell according to claim 10 to regenerate seedlings of the plant, subjecting the seedlings to high temperature treatment at 48° C. for 79 hours, and selecting surviving plants having enhanced high temperature resistance.

13. A polynucleotide comprising the cDNA sequence of SEQ ID NO:2.