# Patent

## Title
Genes for promoting plant growth and use thereof

## Abstract

The invention relates to genes for promoting rapid growth of plant, characterized in genes that are Banana ABC transporter MhPDR1 or MhPDR2 genes, wherein the transporters have amino acid sequences depicted in SEQ ID No: 1 and SEQ ID No: 3, respectively, and the genes have nucleotide sequences depicted in SEQ ID No: 2 and SEQ ID No: 4, respectively. The invention provides further applications of the banana transporter MhPDR1 or MhPDR2 genes, characterized in that the over-expression of the genes in a plant can promote rapid growth of the plant. In addition, the present invention provides a transgenic plant or partial organ, tissue or cells thereof containing the genes or derivatives thereof; as well as provides further a method for promoting rapid growth of a plant.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to genes useful for promoting plant growth, and to the novel use thereof.
2. Description of the Prior Art
Banana (Musa spp.) is a monocotyledon crop of Musaceae, one of the important economic fruit plants, and its growing process is susceptible to physiological stress environment so as to affect its quality, and lower profit of farmer. In view of this, in order to increase the production of banana, the inventor devoted to improve the yield of banana by way of gene transfer so that the improved banana possesses better growing potential, its production period can be shortened, as well as its ability to resist stress environment can be enhanced. ATP-Binding Cassette transporter is a membrane protein commonly exists in organisms. It can hydrolyze ATP to perform active transportation of various substances through biological membrane, such as alkaloids, amino acids, heavy metal chelates, inorganic ion, lipid, peptide and sugars (Higgins, 1992; Biemans-Oldehinkel et al., 2006). Among mammal animals and microorganisms, this gene family had been studied extensively. In plants, however, it is a new research field. Animal study pointed out that ATP-Binding Cassette transporter may participate in the transportation process of drugs, where cancer cells can remove anti-cancer drug out of cell to generate drug resistance. Result in microorganism study indicated that ATP-Binding Cassette transporter was associated with the drug resistance of microorganism. While in plants, ATP-Binding Cassette transporter participated in the transportation process of many substances, and played important role in the mechanisms of growth, development, stress environment resistance and disease resistance of plants (Schulz and Kolukisaoglu, 2006). In this invention, gene transfers of banana ATP-Binding Cassette transporters MhPDR1 (Musa spp. pleiotropic drug resistance 1) and MhPDR2 (Musa spp. pleiotropic drug resistance 2) in a tobacco model plant are utilized as the material, to demonstrate that these genes possess the effect of promoting rapid growth of a plant.
In view of the importance of developing a transferring gene capable of promoting rapid growth of a plant and increasing crop production in the biotechnological industry, the inventor successfully developed genes for promoting rapid growth of plant and uses thereof according to the invention.
SUMMARY OF THE INVENTION
One object of the invention is to provide genes for promoting rapid growth of a plant, characterized in that said genes are selected from banana ATP-Binding Cassette transporter, and promotes the rapid growth of a plant by means of mass expression of said genes.
Another object of the invention is to provide the application of said genes for promoting rapid growth of a plant, characterized in that banana ATP-Binding Cassette transporter gene is transferred in a recombinant plasmid to produce plant cell, organ or transgenic strain containing said recombinant plasmid for application.
Yet another object of the invention is provide a method for promoting rapid growth of a plant, characterized in that mass expression of a banana transporter gene in a plant is carried out to promote the rapid growth of the plant.
Genes for promoting rapid growth of a plant and application thereof that can achieve the above-described objects comprises:
a gene for promoting rapid growth of a plant, comprising at least one selected from the group consisting of banana ATP-Binding Cassette transporter 1 (Mh-PDR1), and banana ATP-Binding Cassette transporter 2 (Mh-PDR2);
wherein said banana ATP-Binding Cassette transporter 1 (Mh-PDR1) has an amino acid sequence as depicted in SEQ ID No: 1 and a nucleotide sequence as depicted in SEQ ID No: 2, and wherein said amino acid sequence in said SEQ ID No: 1 is encoded by the nucleotide sequence in SEQ ID No: 2;
wherein said banana ATP-Binding Cassette transporter 2 (Mh-PDR2) has an amino acid sequence as depicted in SEQ ID No: 3 and a nucleotide sequence as depicted in SEQ ID No: 4, and wherein said amino acid sequence in SEQ ID No: 3 is encoded by the nucleotide sequence in SEQ ID No: 4;
wherein the above-described amino acid sequences include, but not limited to those sequences obtained by mutation, deletion, insertion, or replacement of one to a plurality of amino acid to the sequences depicted in SEQ ID No: 1 or SEQ ID No: 3, while possess the activities of banana ATP-Binding Cassette transporter 1 (Mh-PDR1) or banana ATP-Binding Cassette transporter 2 (Mh-PDR2), as well as the activity of promoting rapid growth of a plant;
wherein the above-described nucleotide sequences include, but not limited to those sequences obtained by mutation, deletion, insertion, or replacement of one to a plurality of nucleotide to the sequences depicted in SEQ ID No: 2 or SEQ ID No: 4, while possess the activity of encoding banana ATP-Binding Cassette transporter 1 (Mh-PDR1) or banana ATP-Binding Cassette transporter 2 (Mh-PDR2), as well as the activity of promoting the rapid growth of a plant.
Term “promoting the rapid growth of a plant” means the nutritive growth, reproductive growth or significantly rapid proliferation, acceleration or increase in the number of cells of a plant.
The above-described term “promoting the rapid growth of a plant” can be further defined as: compared with plant that has not been transferred with the gene described in the invention, the nutritive growth of plant that has been transferred with the gene described in the invention comprises, but not limited to, significant increase on factors such as its root length, plant height, leaf width, while its reproductive growth comprises, but not limited to, factors such as advancing of its blooming time, with its cell number increased significantly also.
In addition to provide gene for promoting rapid growth of a plant, the invention provides further novel application of said gene, includes:
a recombinant plasmid, characterized in that those obtained by constructing banana ATP-Binding Cassette transporter 1 (Mh-PDR1), or banana ATP-Binding Cassette transporter 2 (Mh-PDR2) polynucleotide having open reading frame on the 3′ end of promoter of a vector; wherein said polynucleotide is linked to the 3′ end of said promoter, said promoter can initiate the transcription of said polynucleotide in an organism containing said recombinant plasmid to over-express mRNA of said polynucleotide, and to over-express the protein of said polynucleotide through translation;
wherein the above-described recombinant plasmid or vector comprises, but not limited to: pBI121 (ClonTech), pCAMBIA1301, pCAMBIA1305 (CAMBIA), or other recombinant plasmid or vector suitable to the invention.
Wherein said promoter includes, but not limited to, Cauliflower mosaic virus (CaMV) 35S promoter, or other promoter suitable to the invention.
According to present invention, it can be over-expressing the above-described genes for promoting plant growth individually and singly in a plant, or over-expressing said genes simultaneously in plants.
In addition to provide the above-described gene for promoting rapid growth of a plant, or derivatives thereof, the invention further provides a method for transferring said genes in a plant (banana), and a way for obtaining therefore, but not to limit the obtaining method or source of the invention; the above-described sequences can be obtained by way of chemical or artificial synthesis.
Term “derivative” used therein means various products or combinations containing the above-described genes for promoting the rapid growth of a plant, such as its protein, recombinant plasmid, recombinant protein, pharmaceutical composition, transgenic cell, as well as transgenic plant, or part of organ or tissue of said transgenic plant containing said genes.
The invention provides further a method for promoting the rapid growth of a plant, comprising the following steps:

    step 1: providing cell or tissue of a target plant;
    step 2: transferring the above-described recombinant plasmid containing genes for promoting the rapid growth of a plant into the cell or tissue of the target plant provided in said step 1 to obtain a transgenic plant cell or a transgenic plant tissue; and
    step 3: cultivating said transgenic plant cell or transgenic plant tissue obtained in step 2 to produce a transgenic plant or part of organ, tissue or cell of said transgenic plant containing the above-described recombinant plasmid;


wherein said transferring procedure described in step 2 includes Agrobacterium mediation, genetic recombinant virus infection, transposon vector transferring, gene gun transferring, electroporation, microinjection, pollen tube pathway, liposome mediation, ultrasonic mediation, silicon carbide fiber-mediated transformation, electrophoresis, laser microbeam, polyethylene glycol (PEG), calcium phosphate transferring, or DEAE-dextran-mediated transformation. These features and advantages of the present invention will be fully understood and appreciated from the following detailed description of the accompanying Drawings.
These features and advantages of the present invention will be fully understood and appreciated from the following detailed description of the accompanying Drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic view showing the construction of over-expressed plasmid containing gene for promoting the rapid growth of a plant; FIG. 1A shows the schematic view for constructing MhPDR1; and FIG. 1B shows the schematic view for constructing MhPDR2;
FIG. 2 shows the flow chart for constructing over-expressed plasmid containing gene for promoting the rapid growth of a plant; FIG. 2A: the construction of plasmid pUCRT99gus, FIG. 2B: the construction of plasmid pUCMhPDR1, and FIG. 2C: the construction of plasmid pMhPDR1, FIG. 2D: the construction of plasmid pUCMhPDR2, FIG. 2E: the construction of plasmid pMhPDR2;
FIG. 3 shows the Southern hybridization analytical result of the over-expression of the gene for promoting the rapid growth of a plant in a tobacco transgenic strain; FIG. 3A: construction chart and selection region of probe, FIG. 3B: Southern hybridization analysis indicating that said gene had been inserted entirely into the transgenic tobacco genome, wherein P stands for a probe;
FIG. 4 shows the reverse transcription polymerase chain reaction analysis in a tobacco transgenic strain for over-expression of the gene for promoting the rapid growth of a plant; the control group is a non-transferred strain;
FIG. 4A: the gene expression of Nt-MhPDR1-transferred tobacco,
FIG. 4B: the gene expression of Nt-MhPDR2-transferred tobacco; TRPB2 signal is the control group shows the loading amount of RNA, said gene is a tobacco RNA polymerase subunit (RNA polymerase subunit 2) expressed normally;
FIG. 5 shows the root length difference of four-week old seedlings of tobacco transgenic strain with over-expressed vector containing gene for promoting the rapid growth of a plant; the control group is non-transferred strain; FIG. 5A: the root growing condition of Nt-MhPDR1-transferred seedling; FIG. 5B: comparison of root lengths among Nt-MhPDR1-transferred seedlings; FIG. 5C: root growing conditions of Nt-MhPDR2-transferred seedlings; FIG. 5D: comparison on root lengths among Nt-MhPDR2-transferred seedlings;
FIG. 6 shows external morphologies of over-expressed tobacco transgenic strain Nt-MhPDR1 and Nt-MhPDR2 with vector containing gene for promoting the rapid growth of a plant; the control group is a non-transferred strain; FIG. 6A: Nt-MhPDR1 one week after subculturing; FIG. 6B: Nt-MhPDR2 one week after subculturing; FIG. 6C: 3-month old Nt-MhPDR1; FIG. 6D: 3-month old Nt-MhPDR2;
FIG. 7 shows the growth survey of over-expressed tobacco transgenic strain with vector containing gene for promoting the rapid growth of a plant; the control group is a non-transferred strain; FIG. 7A: the height change of Nt-MhPDR2 transgenic strain; FIG. 7B: the leaf width change of Nt-MhPDR2 transgenic strain;
FIG. 8 shows the advancing of blooming of over-expressed tobacco transgenic strain with vector containing gene for promoting the rapid growth of a plant; FIG. 8A: the blooming condition of 8-month old Nt-MhPDR1 transgenic strain; the control group (non-transferred strain) did not bloom yet; FIG. 8B: the blooming condition of 8-month old Nt-MhPDR2 transgenic strain, the control group (non-transferred strain) did not bloom yet;
FIG. 9 shows the observation result of gas hole in over-expressed tobacco transgenic strain with vector containing gene for promoting the rapid growth of a plant; FIG. 9A: non-transferred strain (the control group); FIG. 9B is the Nt-MhPDR1-1 transgenic strain; FIG. 9C is the Nt-MhPDR2-1 transgenic strain; FIG. 9D is the Nt-MhPDR2-2 transgenic strain; FIG. 9E is the Nt-MhPDR2-3 transgenic strain; FIG. 9F is the Nt-MhPDR2-4 transgenic strain; FIG. 9G is the Nt-MhPDR2-5 transgenic strain; FIG. 9H is the Nt-MhPDR2-6ransgenic strain; Bar=20 μm;
FIG. 10 shows the comparison of lengths of guard cells among over-expressed tobacco transgenic strains with vectors containing gene for promoting the rapid growth of a plant, no significant difference had been observed; wherein 1-1 represents Nt-MhPDR1-1 transgenic strain, 2-1 represents Nt-MhPDR2-1 transgenic strain; 2-2 represents Nt-MhPDR2-2 transgenic strain; 2-3 represents Nt-MhPDR2-3 transgenic strain; 2-4 represents Nt-MhPDR2-4 transgenic strain; 2-5 represents Nt-MhPDR2-5 transgenic strain; and 2-6 represents Nt-MhPDR2-6 transgenic strain.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The invention will be illustrated in more details with the following examples; however, the invention is not limited by those examples.
Example 1
The Transfer of Banana ATP-Binding Cassette Transporter Genes for Promoting Rapid Growth of a Plant
1. Source of Banana λEMBL3 Genomic Library
Banana genomic library was obtained by extracting genomic DNA of a banana variety, Hsien Jin Chiao, Musa spp., AAA group, plant leave, and then, by using bacteriophage λEMBL3 as the vector, genomic library was constructed by means of cleavage replacement of DNA fragment.
2. Preparation and Labeling of Nucleic Acid Probe
A gene fragment of Arabidopsis thaliana ecotype Columbia ETR1 gene (GeneBank accession number NP—176808) was used as the template to carry out the preparation of a nucleic acid probe by using Prime-A-Gene kit (Promega, USA) according to the following process: total reaction volume was 50 μL, comprising: 1× labeling buffer, pH6.6 {50 Mm Tris-HCL, pH8.3, 5 mM MgCl2, 2 mM DTT, 0.2M HEPES [N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)], 26A260 unit/mL random hexadeoxyribonicleotides}, 20 μM dATP, dGTP, dTTP, 500 ng/mL denatured DNA template, 400 μg/mL Bovine serum albumin (BSA), 50μ Ci [α-32P] dCTP (333 nM), and 5 unit Klenow DNA Polymerase. After reacting at 37° C. for 2 hours, 2 μL 0.5M EDTA (pH 8.0) was added to terminate the reaction, followed by adding 8 μL tracing dye (50% glycerol, 0.25% bromophenol blue), and then the reaction solution was passed through Sephadex-G50 chromatograph column, eluted with TE (pH7.6) buffer solution, every 160 ̃180 μL was collected in a tube, and after determining radioactivity of every tube in a liquid scintillation counter (Liquid Scintillation Counter, Beckman 1801), suitable amount of eluate with highest radioactivity was used as the probe.
3. Genomic Library Screening of Banana ATP-Binding Cassette Transporter Gene Mh-PDR
Banana genomic library was screened by plaque hybridization. At first, E. coli strain XL1-Blue MRA (P2) was used as the infection host of λEMBL3, and was cultured in a NZY medium (every liter containing 5 g NaCl, 2 g MgSO4.7H2O, 5 g yeast extract), and total of 150 million plaque forming units were screened under high stringency.
A nitrocellulose membrane was used to transfer bacteriophage. The transfer membrane was treated with denature buffer (0.5M NaOH, 1.5M NaCl) for 2 minutes, then treated with neutralization buffer [0.5M Tris base, 1.5M NaCl, 0.035% HCl (v/v)] for 5 minutes, and finally, immersed in a 2×SSPE (1×SSPE 0.18M NaCl, 10 mM NaH2PO4, 1 mM EDTA pH7.4) for 30 seconds. Thereafter, it was treated in an 80° C. vacuum oven for 2 hours to fix the bacteriophage DNA. Then, it was placed in a solution containing 2×SSPE and 0.1% SDS, and was shaken mildly at room temperature for one hour. Then, placed the nitrocellulose membrane in a pre-hybridization solution containing 5×SSPE, 5×BFP (1×BFP containing 0.02% BSA, 0.02% Ficoll-400000, 0.02% PVP-360000), 0.1% SDS, 50% formamide and 500 μg/mL salmon sperm DNA, and carried out pre-hybridization reaction at 37° C. for 2 hours. Thereafter, the radio-labeled probe was used to carry out hybridization reaction in 5×SSPE, 1×BFP, 0.1% SDS, 50% formamide and 100 μg/mL salmon sperm DNA with the membrane at 37° C. for 16 ̃18 hours. Then, the nitrocellulose membrane was treated with wash buffer I (5×SSPE, 0.1% SDS) twice at room temperature for 15 minutes, as well as twice with wash buffer II (1×SSPE, 0.5% SDS) at 37° C. for 15 minutes to wash off non-specific probe. After developed by press exposure with X-ray film at −80° C. (Kodak XAR film), the bacteriophage containing target gene DNA could be detected. Said bacteriophage was isolated from the medium, kept in a SM buffer solution containing 0.03% chloroform, and was purified several times to obtain genomic clone of banana ATP-Binding Cassette transporter.
4. Extraction of Selected Bacteriophage Clone DNA
The bacteriophage liquor of the target clone was cut open with toothpick in a NZY solid medium, 3 mL Top agar containing host cell of E. coli XL1-Blue MRA (P2) was added, and was cultivated in the NZY solid medium at 37° C. for 8 hours. On the next day, a capillary was used to poke up single plaque agar piece on the cut line, and was cultured by spreading over a NZY solid medium at 37° C. for 7 ̃11 hours. Then the medium was placed in a 4° C. refrigerator, SM buffer (each liter containing 5.8 g NaCl, 2 g MgSO4.7H2O, 50 ml 1 M Tris-HCl (pH 7.5), 0.1 g gelatin) was added to dissolve off the bacteriophage. The solution was collected in a centrifuge tube, chloroform was added to 0.03%, and was centrifuged at 4° C. and 7,000 rpm (Beckman J2-MC, JS-13.1) for 5 minutes, and was kept at 4° C. for use later. Thereafter, the above-described mass reproduced target clone bacteriophage was used to infect host cells at a cell count ratio of 5:1. after mixed with 1 mL SM buffer solution and 2.5 mM CaCl2 5 mL, the solution was stood still at room temperature for 15 minutes, and then at 37° C. for 45 minutes. Thereafter, 100 mL 2×NZY liquid medium (0.4% MgSO4.7H2O, 2% NaCl, 1% bacto-yeast extract, 2% NZ amine, 0.2% casaimino acid, 5 mM MgSO4, 25 mM Tris-HCl pH7.5) was poured in and was cultured by shaking at 37° C. and 240 rpm for more than 8 hours. 4.5 mL Chloroform was then added and treated by shaking at 37° C. and 240 rpm for 15 minutes. The suspension was then centrifuged at 4° C. and 7,000 rpm for 20 minutes (Beckman J2-MC, JA 10 rotor). To the supernatant, 100 μL DNase I (1 mg/mL) and 100 μL RNaseA (10 mg/mL) was added and treated at 37° C. and 80 rpm for 45 minutes. Then, 33 mL 4M NaCl was added and was treated in an ice bath for 1 hour. Thereafter, 33 mL ice-cold 50% polyethylene glycol was added, and precipitated at 4° C. overnight. The suspension was centrifuged at 4° C. and 5,000 rpm for 20 minutes (Beckman J2-MC, JA 10 rotor). The supernatant was removed, and the residue was air dried. 500 μL PKB solution (10 mM NaCl, 10 mM Tris-HCl pH8.0, 10 mM EDTA, 0.1% SDS) was added to re-suspend the precipitate. Then, proteinase K (final concentration: 12.5 μg/mL) was added and reacted at 37° C. for 20 minutes. This was extracted in turn with equal volume of phenol, PCI (phenol:chloroform:isoamyl alcohol=25:24:1), and CI (chloroform:isoamyl alcohol=24:1). After centrifuged at room temperature and 14,000 rpm for 5 minutes, 2-fold volume of −20° C. 100% ethanol was added to the supernatant, and centrifuged at 4° C. and 14,000 rpm for 10 minutes. The supernatant was decanted, and the residue was air dried. The precipitated DNA was washed with 70% ethanol and 100% ethanol, respectively. It was dissolved in TE buffer solution (pH7.5) and stored at 4° C. for later use.
5. Sequencing of DNA
An automatic nucleic acid sequencer (ABI sequencer 377) was used to carry out the sequencing of DNA to obtain sequences of Mh-PDR genomic clone of banana ATP-Binding Cassette transporter gene.
6. Establishment of Complementary DNA Library (cDNA Library)
Total banana RNA was extracted at first as followed: plant material was cut and was ground in liquid nitrogen into powder. 20 mL 65° C. Extraction buffer (2 M NaCl, 25 mM EDTA, pH 8.0, 100 mM Tris-HCl, spermidine 0.5 g/L, 3% Hexadecyl trimethyl-ammonium bromide, 3% polxviuyl-pyrrolidone-40, 0.4% 2-mercaptoethanol) was added, stirred in a homogenizer, and treated at 65° C. for 10 minutes. Equal amount of CI (chloroform:isoamyl alcohol=49:1) was added, mixed and centrifuged. The supernatant was re-extracted once more, and 1⁄3-fold volume of 8 M LiCl was added thereto. After precipitated at 4° C. overnight, it was centrifuged at 4° C. The supernatant was discarded, and the RNA residue was suspended in 0.5% SDS. Equal volume of CI was added and mixed by shaking for several seconds. After centrifuged at 4° C., 2-fold volume of 100% ethanol was added into the supernatant, and the mixture was precipitated at −20° C. Thereafter, it was centrifuged at 4° C. to discard the supernatant. 500 μL of 70% ethanol was added, centrifuged at 4° C. to discard the supernatant. Then, 500 μL of 100% ethanol was added, centrifuged at 4° C. to discard the supernatant. The RNA precipitate was air dried. The RNA was then dissolved in a suitable amount of DEPC water, the concentration was determined and then stored for later use. Finally, complementary DNA library was completed by using λZAP cDNA synthesis kit provided by Stratagene.
7. Screening of Complementary DNA Library
In order to obtain the cDNA sequence of banana ATP-Binding Cassette transporter gene, a fragment of the genomic clone was used as the probe, plaque hybridization was used to screen cDNA library of Musa spp. Hsien Jin Chiao, AAA group, and obtained two type of ATP-Binding Cassette transporter cDNA clone. Of these, pMaABC-29 cDNA had a nucleotide length of 4731 bp (SEQ ID No: 2), which could translate a protein having a total length of 1452 amino acids (SEQ ID No: 1), a pre-estimated molecular weight of 163 kDa, an isoelectric point of 8.20. Its corresponding gene was named as Mh-PDR1; and clone pMaABC-74p cDNA had a nucleotide length of 4760 bp (SEQ ID No: 4), could translate a protein having a total length of 1468 amino acids (SEQ ID No: 3), a pre-estimated molecular weight of 165 kDa, and isoelectric point of 8.50. Its corresponding gene was named as Mh-PDR2. The identity of the amino acid sequence between these two clones was 85.3%.
Example 2
Construction of Vector Containing Gene for Promoting the Rapid Growth of a Plant and its Gene Transfer
Vector containing gene for promoting the rapid growth of a plant was carried out as described in FIG. 1 under a flow scheme as followed:
(1) Material for Plasmid


    a. pRT99gus plasmid, having a total length of 6.7 Kb, containing a GUS reporter gene under control of CaMV 35S promoter, and a NOS terminator (GenBank accession no. L09137).
    b. pGKU plasmid, having a total length of 7.4 Kb, containing a NPTII plant screening gene under control of CaMV 35S promoter, a GUS reporter gene under control of CaMV 35S promoter and a NOS terminator.

(2) Construction Flow Scheme

Step 1: Construction Strategy of pUCRT99gus



pUC19 was cleaved at first with EcoRI and SalI to remove large part of MCS region on the pUC19, the then blunt treated with Klenow enzyme, and subjected to electrophoresis separation to recover a 2.6 kb fragment. This fragment was subjected to self-ligation to obtain an intermediate vector pUC19m. This intermediate vector pUC19m was cleaved with restriction enzyme HindIII to recover a fragment of about 2.6 kb in length. A plasmid pRT99gus was cleaved completely with HindIII to recover a DNA fragment of 2.6 Kb that was used as the DNA fragment to be inserted. These two fragments were subjected to ligation to obtain a plasmid having total length of 5.2 Kb, which was named as pUCRT99gus (FIG. 2A).
Step 2: Construction Strategy of Plasmid pMhPDR1
Banana total RNA treated with DNase was subjected to reaction by using One-Step RT-PCR Kit (GeneMark), wherein the reaction solution contained, 0.1 μg/μL template RNA, 50 ng/μL primers, 1× Reaction Mix, 1× Enhancer, 2% Enzyme Mix. The reaction temperature was 50° C. 30 minutes, 94° C. 2 minutes, then 35 cycles of 94° C. 30 seconds, 59° C. 30 seconds, and 72° C. 1 minute. Finally, reacted at 72° C. for 10 minutes, stored at 4° C. for later use. Primers used included:







forward primer PDR1-1: 


(contained XbaI restriction site, total 27 mer)








5′-tcc gaaccgagcgaggtg-3′ 
(SEQ ID No: 5)







       XbaI Met


 


reverse primer PDR1-2: 


(contained KpnI restriction site, total 28 mer)








5′-agc tcatctcttctggaagttg-3′
(SEQ ID No: 6)







       KpnI






Gene fragments obtained in the above-described reverse transcription polymerase chain reaction were cleaved completely with XbaI and KpnI to recover a DNA fragment of 4.8 Kb, which was used as the DNA fragment to be inserted. pUCRT99gus plasmid was cleaved completely with XbaI and KpnI to recover a DNA fragment of 3.4 Kb, which was used as the vector DNA fragment. These two fragments were subjected to ligation to obtain a plasmid having a total length of about 8.2 Kb, which was named as pUCMhPDR1 (FIG. 2B). Plasmid pUCMhPDR1 was cleaved completely with SphI to recover a DNA fragment of 5.3 Kb, which was used as the DNA fragment to be inserted. Plasmid pGKU was cleaved completely with SphI to recover a DNA fragment of 7.4 Kb, which was used as a vector DNA fragment. These two fragments were subjected to ligation to obtain a plasmid having a total length of 12.89 Kb, which was named as pMhPDR1 (FIG. 2C).
Step 3: Construction Strategy of Plasmid pMhPDR2
Banana total RNA treated with DNase was subjected to reaction by using One-Step RT-PCR Kit (GeneMark), wherein the reaction solution contained, 0.1 μg/μL template RNA, 50 ng/μL primers, 1× Reaction Mix, 1× Enhancer, 2% Enzyme Mix. The reaction temperature was 50° C. 30 minutes, 94° C. 2 minutes, then 35 cycles of 94° C. 30 seconds, 59° C. 30 seconds, and 72° C. 1 minute. Finally, reacted at 72° C. for 10 minutes, stored at 4° C. for later use. Primers used included:







forward primer PDR2-1: 


(contained XbaI restriction site, total 27 mer)








5′-tcc gagccgagcgaggtg-3′
(SEQ ID No: 7)







       XbaI Met


 


reverse primer PDR2-2:


(contained KpnI restriction site, total 28 mer)








5′-agc tcatctcttttggaagttg-3′
(SEQ ID No: 8)







       KpnI






Gene fragments obtained in the above-described reverse transcription polymerase chain reaction were cleaved completely with XbaI and KpnI to recover a DNA fragment of 4.7 Kb, which was used as a DNA fragment to be inserted. Plasmid pUCRT99gus was cleaved completely with XbaI and KpnI to recover a DNA fragment of 3.4 Kb, which was used as a vector DNA fragment. These two fragments were subjected to ligation to obtain a plasmid having a total length of about 8.1 Kb, which was named as pUCMhPDR2 (FIG. 2D). Plasmid pUCMhPDR2 was cleaved completely with SphI to recover a DNA fragment of 5.3 Kb, which was used a DNA fragment to be inserted. Plasmid pGKU was cleaved completely with SphI to recover a DNA fragment of 7.4 Kb, which was used as a vector DNA fragment. These two fragments were subjected to ligation to obtain a plasmid having a total length of about 12.8 Kb, which was named as pMhPDR2 (FIG. 2E).
2. Agrobacterium Transformation by Electroporation
Agrobacterium cell to be transformed was thawed on ice, 100 ng plasmid DNA was mixed well thereto on an ice-water bath, and placed in an electroporation cuvette. After electric shock in an Electro Cell Manipulator 200 (BTX®) with a voltage of 1420 V, 0.5 mL YEB liquid medium was added, cultured at 28° C. for 1 hour, then placed in a solid medium containing suitable antibiotics, and cultured at 28° C. for 48 hours.
3. Agrobacterium Mediated Gene Transfer
A single colony of Agrobacterium with vector construct containing gene for promoting the rapid growth of a plant in 20 mL YEB liquid medium containing antibiotics (each liter containing 5 g Beef extract, Yeast 1 g extract, 5 g Peptone, 5 g Mannitol, 0.5 g MgSO4), and cultured by shaking at 28° C. and 240 rpm for 48 hours. The bacteria liquor was diluted to OD600=0.8. 20 mL bacteria liquor was placed in a glass Petri dish. A tobacco leaf was cut in the bacteria liquor into round leaf piece of 1.5 cm×1.5 cm, which round leaf pieces were placed on N01B1 solid medium [MS, 0.1 mg/L1-naphthyl acetic acid, 1 mg/L BA, 3% sucrose, pH5.7, 0.7% agar], and cultured at 25° C., 16 hour-lighting environment for 3 days. These round leaf pieces were then placed on a N01B1 solid medium containing 250 mg/L cefotaxime and 100 mg/L kanamycin, and screened by culturing at 25° C. and 16-hour lighting environment for about two weeks. Those round leaf pieces with grown shoot were placed on N01B1 solid medium containing 250 mg/L cefotaxime and 200 mg/L kanamycin, and sub-screened by cultured at 25° C. and 16-hour lighting environment. Until the shoot had grown to about 1 cm in length, non-whitened shoots could be cut off, cottage on a MS solid medium containing 250 mg/L cefotaxime and 200 mg/L kanamycin, and cultivated at 25° C. and 16-hour lighting environment to develop into a plant.
Example 3
Molecular Assay on Tobacco Transferred with Gene for Promoting the Rapid Growth of a Plant Using Southern Hybridization Assay
This example demonstrated the gene integrity after inserting transfer gene into genome of tobacco transgenic strain by using Southern hybridization assay, which comprised of cleaving tobacco transgenic strain with restriction enzymes, using target gene as the probe and carrying out hybridization assay.
1. Extraction of Transgenic Strain Genomic DNA
Leaves of transgenic strain were quick frozen in liquid nitrogen, 1 g of the leave was placed in a mortar and ground into powder. 15 mL extraction buffer (100 mM Tris-HCl, pH 8.0, 50 mM Na2EDTA, pH 8.0, 500 mM NaCl, 100 mM 2-mercaptoethanol) was added thereto and mixed well. 1 mL 20% SDS was then added and stood still at 0° C. for 20 minutes. Next, it was centrifuged at 8,800 rpm and 4° C. for 30 minutes (Beckman J2-MC, JA-18 rotor). The supernatant was filtered through 100 meshes Nylon mesh into 10 mL-20° C. isopropanol contained in a centrifuge tube, and stood still at −20° C. for 30 minutes. Then, centrifuged at 8,800 rpm and 4° C. for 30 minutes to discard the supernatant. 0.7 mL 4° C. High-TE buffer solution (50 mM Tris-HCl, pH 8.0, 50 mM Na2EDTA) was added to the residue to dissolve the product, which was centrifuged at 4° C. and 13,200 rpm for 10 minutes. The supernatant was removed to a new centrifuge tube, 75 μL 3 M sodium acetate (pH 5.2) and 500 μL isopropanol was added thereto. After mixed homogeneously, it was stood still at −20° C. for 30 minutes. Then, it was centrifuged at 13,200 rpm and 4° C. for 30 minutes to discard the supernatant. After salt washed with 70% ethanol and 100% ethanol, 100 μL TE buffer solution (10 mM Tris-HCl, pH 8.0, 1 mM Na2EDTA) and suitable amount of RNaseA were added, and reacted at 37° C. for 30 minutes, stored at 4° C. for later use.
2. Preparation of Nucleic Acid Probe and Southern Hybridization Analysis
The synthesis and labeling of probe was carried out by using Primer-a-gene kit (Promega). After reacting at 37° C. for 1 hour, 2 μL 0.5M Na2EDTA was added to terminate the reaction, and 8 μL tracing dye (50% glycerol, 25% bromophenol blue) was added also. The reaction solution was passed through Sephadex-G50 chromatograph column, and the synthetic material was eluted off with TE buffer solution (pH 7.6), which was counted in a liquid scintillation counter (Liquid Scintillation Counter; Beckman 6501) to determine the radioactivity, and several collection tubes of elution solution with highest activity were used as the probe.
20 μg transgenic strain genomic DNA was cleaved with restriction enzyme BamHI, and separated by electrophoresis on 0.7% agarose gel. The gel was soaked successively in 2.5 N HCl, denature buffer solution (1.5 M NaCl, 0.5 M NaOH), and neutralization buffer solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0) each for 15 minutes, and the process was repeated twice. The gel thus-treated was DNA transferred on Hybond N transfer membrane, and cross-linked under condition of UV 120 mJ/cm2 (Spectrolinker XL-1500), which was then placed in a 80° C. vacuum oven for 1 hour to immobilize DNA. Thereafter, the transfer membrane was soaked at first in a pre-hybridization solution (6×SSPE, 1% SDS, 5×BFP, 50 μg/mL salmon sperm DNA) and treated at 65° C. for more than 2 hours. Next, the transfer membrane was soaked in hybridization solution containing the above-described probe (6×SSPE, 0.5% SDS, 5×BFP, 250 μg/mL salmon sperm DNA, 10% dextran sulfate), and treated at 65° C. for 16-18 hours. It was shaken in a wash solution I (2×SSPE, 0.1% SDS) at room temperature for 15 minutes, and this process was repeated twice. Then, it was shaken mildly in a wash solution II (1×SSPE, 0.1% SDS) at 65° C. for 15 minutes, and this process was repeated twice. Finally, it was pressed exposure on a X-ray film at −80° C.
3. The Result of Southern Hybridization Analysis
This example used MhPDR2 as the illustrative example to compare the nucleotide sequence of MhPDR2 decoding region and of the tobacco ATP-Binding Cassette transporter gene, banana MhPDR2 gene had an identity of 68.2% against tobacco NtPDR1 (GenBank accession no. AB075550); and an identity of 67.9% against tobacco NtPDR2 gene (GenBank accession no. AB109389). Therefore, region with low identity was selected as the probe 585 bp used in Southern hybridization analysis (FIG. 3A) to carry out hybridization. The result of hybridization was shown in FIG. 3B. All of transgenic strain Nt-MhPDR2-2 ̃6 yielded hybridization signals at site of the predicted target fragment 5.2 Kb. This demonstrated that said gene for promoting rapid growth had been transferred integrally and successfully in the tobacco genome. In addition, both non-transgenic strain and transgenic strain Nt-MhPDR2-1 ̃6 yielded hybridization signals at 1.4 Kb, 2.4 Kb, 3.0 Kb, 9.0 Kb and 12.0 Kb, which was postulated to be tobacco endogenous homogeneous similar genes.
Example 4
Gene Expression Assay of Tobacco Transferred with Gene for Promoting the Rapid Growth of a Plant
This Example assayed the expression of transferred gene in tobacco by using reverse transcription polymerase chain reaction. The results indicated that after transferring gene for promoting the rapid growth of a plant into tobacco, gene expression could be carried out readily, and products of the gene for promoting the rapid growth of a plant could be accumulated.
1. Extraction of Transgenic Strain RNA
Material frozen in liquid nitrogen was ground in a mortar with liquid nitrogen to break the plant tissues. To each gram of the powdered tissue, 2-3 mL solution D without sarcosyl [4 M guanidiun thiocyanate, 25 mM sodium citrate (pH7.0), 0.1 M β-mercaptoethanol], and equal volume of  PCI (phenol:chloroform:isoamyl alcohol=25:24:1) were added and mixed well in a homogenizer. More sarcosyl was added to a final concentration of 0.5%, which, after mixing in a homogenizer, was centrifuged at 4° C. and 10800 rpm for 20 minutes (Beckman J2-MC, JS 13.1). The supernatant was extracted once with equal volume of PCI, and finally, equal volume of CI (chloroform:isoamyl alcohol=49:1) was used to centrifuge extraction of the supernatant. Volume of the supernatant was determined, 1/10 volume of 3 M NaOAc (pH5.2) and 2.5-fold volume of −20° C. 100% ethanol were added, which was shaken homogeneously and precipitated at −80° C. overnight. On the next day, it was centrifuged at 4° C. and 10800 rpm for 15 minutes. The residue was desalted with 2 mL 70% ethanol and 100% ethanol, respectively, under the condition of centrifuging at 4° C. and 10800 rpm for 5 minutes. The supernatant was discarded, and the RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water. LiCl was added to the final concentration of 2.5 M. Then, 1% β-mercaptoethanol was added and the mixture was precipitated at −80° C. overnight. On the third day, it was centrifuged at 10800 rpm and 4° C. for 90 minutes, desalted with 70% ethanol and 100% ethanol, respectively. The RNA was air dried, dissolved in DEPC water, and determined quantitatively by measuring its OD260.
2. Demonstration of Gene Expression by Reverse Transcription Polymerase Chain Reaction
Tobacco total RNA treated with DNase was subjected to reaction by using One-Step RT-PCR Kit (GeneMark), wherein the reaction solution contained 0.1 μg/μL template RNA, 50 ng/μL primers, 1× Reaction Mix, 1× Enhancer, 2% Enzyme Mix. The reaction temperature was 50° C. 30 minutes, 94° C. 2 minutes, and then 40 cycles of 94° C. 30 seconds, 57.5° C. 30 seconds, and 72° C. 1 minute. Finally, it was reacted at 72° C. for 10 minutes, and stored at 4° C. for later use. Transgenic strain Nt-MhPDR1 and Nt-MhPDR2 used the following primers:







forward primer cABC1: total 16 mer








5′-atggagccgagcgagg-3′
(SEQ ID No: 9)


 


reverse primer 74P-F3: total 19 mer



5′-ggcctctagcatgttaagg-3′
(SEQ ID No: 10)






Result of RT-PCR was shown in FIGS. 4A and 4B, wherein wild type tobacco (the control group) yielded no signal, while both transgenic strain Nt-MhPDR1 and Nt-MhPDR2 yielded predicted 0.46 kb hybridization signal. This indicated that both of these two genes could be over-expressed in transgenic strain under the activation of CaMV35S promoter, and could accumulate the product of the gene for promoting the rapid growth of a plant.
Example 5
The Transferred Gene could Promote Effectively the Nutritive Growth of the Transgenic Tobacco
In order to observe external morphologies of transgenic strain Nt-MhPDR1 and Nt-MhPDR2, this Example performed two variation analysis: (1) root length of transgenic seedlings; and (2) height and leaf width increase of transgenic plant, and the results indicated that the transferred gene could promote effectively the nutritive growth of transgenic tobacco.
1. The Transferred Gene Could Promote Effectively the Root Growth of Transgenic Tobacco Seedlings
Seeds of R2 generation tobacco transferred with gene for promoting rapid growth were cultivated in MS medium, and the medium was stood upright during the cultivation period. After four weeks, they were observed and their root lengths were measured. The results were shown in FIG. 5A to 5D. The observation of the growth condition of transgenic strain R2 generation seedlings revealed that, the average root length of the control group seedlings was 1.2 cm; average root length of Nt-MhPDR1 seedlings was 5.0 cm; and average root length of Nt-MhPDR2 seedlings was 4.4 cm. These remarkable differences demonstrated that the transferred gene could promote effectively the root growth of transgenic tobacco seedlings.
2. The Transferred Gene could Promote Effectively the Growth of Height and Leaf Width of Transgenic Tobacco
Shoot apical meristem of transgenic strains Nt-MhPDR1 and Nt-MhPDR2 were cultivated in MS medium for 4 weeks, and then they were removed out of the bottle for growth variation analysis. The analytical method comprised of observing and recording their plant height and leaf width every week, wherein plant height was recorded as the measurement of the distance from ground surface to the growing point, while leaf width was measured as followed: the greatest width of the leaf was measured as X axis, then other width was measured as Y axis, the leaf width was calculated as [(X axis+Y axis)/2], the seedlings were removed out of the bottles after cottage for 4 weeks.
Results indicated that, compared with the control group, the growing potential of transgenic strain was better, as shown in FIG. 6A to 6D. Growing survey on Nt-MhPDR2 was further carried out, wherein plant height survey was shown in FIGS. 7A and 7B. On one week after removed out of the bottle, the average plant height of the control group was 2.2 cm; while the average plant height of MhPDR2 was 3.71 cm. The observation on the seventh week indicated that the average plant height of the control group was 11.3 cm; while the average plant height of MhPDR2 was 27.6 cm, and the average plant height difference was 16.3 cm. The survey result of leaf width was shown in FIG. 7B. On one week after removed out of the bottle, the average leaf width of the control group was 5.1 cm; the average leaf width of MhPDR2 was 11.0 cm. The observation on the seventh week indicated that, the average leaf width of the control group was 27.2 cm; while the average leaf width of MhPDR2 was 48.5 cm, and the leaf width difference was 21.3 cm. The plant height and leaf width had been subject to statistical analysis by Student's t-test, and all of the results indicated significant difference (*P<0.05). These demonstrated that the transferred gene could promote the nutritive growth of the transgenic tobacco.
Example 6
Transferring Gene for Promote Rapid Growth could Advance the Blooming of the Transgenic Tobacco
In order to observe the reproductive growth conditions of transgenic strain Nt-MhPDR1 and Nt-MhPDR2, this Example illustrated with figures that the transferred gene for promote rapid growth could promote, not only the nutritive growth rate such as root growth rate during seedling period, and plant height and leaf width of tobacco, but also could shorten time needed for blooming to advance into reproductive growth period. Source of plant material of non-transgenic strain was same as those of transgenic strain clone that was subjected early to Agrobacterium transfer. Plant material of non-transgenic strain was subjected to callus tissue induction of round leaf pieces simultaneously with transgenic strain, but not subjected to antibiotic screening. New buds were removed onto same medium to differentiate into full plants, which were removed out of the sterile bottles simultaneously with transgenic strain to carry out domestication and planting. Results were shown in FIGS. 8A and 8B, which revealed that transgenic tobacco transferred with gene for promoting the rapid growth of a plant could advance blooming time remarkably. On the eighth month, the inflorescence of the transgenic strain began to wither, and its seeds were ripened, while non-transgenic strain did not bloom yet.
Example 7
The Mechanism of the Gene for Promoting Rapid Growth
In order to define clearly whether the rapid growth of a plant caused by the gene for promoting the rapid growth of a plant was induced by the enlarging of cell volume or not, the size of cell was further observed. Tobacco was cut into leaf pieces of 0.5 cm×0.5 cm. Phosphate buffer (each liter containing 39 mL 0.2 M monobasic sodium phosphate, 61 mL 0.2 M dibasic sodium phosphate, pH 7.0) was used to formulate 2.5% Glutaraldehyde as immobilizing solution. Leaf pieces were immobilized in the immobilizing solution for 4 hour. The immobilizing solution was removed and phosphate buffer was added. The replacement was repeated for 6 times. Leaf pieces were fixed in 2% OsO4 at 4° C. for 3 hours. The supernatant was discarded, and 30%, 50%, 70%, 80%, 95%, 100% were added successively to dehydrate. The supernatant was discarded, 100% acetone was added to carry out post-treatment. Leaf pieces were removed and dried by critical point drying (CPD). An aluminum stage sample made through gold cladding was placed in a S-3000N scanning electro microscope, and performed the scanning observation under an acceleration voltage of 15 KV.
The observation result of scanning electro microscope was shown in FIG. 9A to 9H, and FIG. 10. The average length of the control group guard cell was 29.45 μm, the average length of transgenic strain MhPDR1-1 guard cell was 29.45 μm, and the average length of transgenic strain MhPDR2-1 ̃6 guard cell was 32.3, 30.4, 27.5, 32.3, 28.5 and 26.25 μm, all of these data did not show significant difference. Therefore, the better plant height and leaf width of transgenic strain tobacco containing gene for promoting rapid growth did not come from the enlarging of cell volume, but due to the increase of the whole number of cells.
Accordingly, it could be postulated that MhPDR gene might participated in the control of mechanisms associated with the growth and development of a plant, and might affect the growth and development through the control of nutritional elements, and allowed the growth potential of the transgenic strain become better than that of non-transgenic strain.
Many changes and modifications in the above described embodiments of the invention can, of course, be carried out without departing from the scope thereof. Accordingly, to promote the progress in science and the useful arts, the invention is disclosed and is intended to be limited only by the scope of the appended claims.

## Claims

1. An isolated polynucleotide for promoting the rapid growth of a plant comprising a nucleic acid sequence encoding an ATP-binding cassette transporter comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:3.

2. The polynucleotide of claim 1, wherein said polynucleotide comprises SEQ ID NO: 2 or SEQ ID NO: 4.

3. A recombinant plasmid comprising a nucleic acid sequence operably linked to the 3′ end of a promoter, wherein said nucleic acid sequence encodes an ATP-binding cassette transporter comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:3.

4. A plant, or part thereof, organ, tissue or cell of said plant containing the recombinant plasmid as recited in claim 3 constructed by gene transfer.

5. A method for promoting the rapid growth of a plant, comprising the following steps:
step 1: providing a cell or tissue of a target plant;
step 2: transforming the recombinant plasmid as recited in claim 3 into the cell or tissue of the target plant provided in step 1 to obtain a transgenic cell of said plant or a transgenic tissue of said plant; and
step 3: cultivating the transgenic cell or transgenic tissue of said plant obtained in step 2 to produce a transgenic plant or part thereof, organ, tissue or cell of said transgenic plant containing the recombinant plasmid as recited in claim 3.

6. The method as recited in claim 5, wherein said transforming method described in step 2 comprises one selected from the group consisting of Agrobacterium mediation, genetic recombinant virus infection, transposon vector transferring, gene gun transferring, electroporation, microinjection, pollen tube pathway, liposome mediation, ultrasonic mediation, silicon carbide fiber-mediated transformation, electrophoresis, laser microbeam, polyethylene glycol (PEG), calcium phosphate transferring, and DEAE-dextran mediated transformation.