# Patent

## Title
Leaf-specific chlorophyll a/b binding protein gene promoter from oil palm

## Abstract

The present invention discloses a promoter sequence of light-harvesting chlorophyll a/b binding protein pGWLS01 isolated from the oil palm leaf. This promoter enables the manipulation of oil palm leaves for the production of high value-added products via genetic engineering tools. The novel features of the promoter itself which regulate high and specific expression of foreign genes in the leaves will avoid the interference of novel products in the commodity oil extracted from mesocarp and kernel tissues. Furthermore, the promoter is also potentially useful in the production of insect-resistant palm.

## Background
TECHNICAL FIELD OF THE INVENTION
The invention is related to type I chlorophyll a/b binding protein gene with abundant and specific expression in the leaf of the Palmae family, its regulatory sequence, and the use of its regulatory sequence for controlling the expression of foreign genes to produce high value-added products in the leaves of transgenic plants.
SUMMARY OF THE INVENTION
The present invention relates to the promoter sequence of the light-harvesting chlorophyll a/b binding protein gene pGWLS01 which was isolated from the oil palm genome. The presence of this promoter sequence will enable the manipulation of oil palm leaves for producing high value-added product via the introduction of foreign genes into the oil palm genome using genetic engineering tools. Furthermore, the promoter is also potentially useful in the production of insect resistant palm for the purpose of crop protection. The novel features of the promoter itself which regulate high and specific expression of foreign genes in the leaves will avoid the interference of novel products in the commodity oil extracted from mesocarp and kernel tissues. The presence of leaves throughout the plant life cycle will also enable early harvesting and continuous supply of novel metabolites.
Three different approaches (RT-PCR, cDNA library screening and 5′-RACE) were employed in the isolation of cDNA that encodes for the light-harvesting chlorophyll a/b binding protein gene (LS01). RT-PCR and screening of leaf cDNA library resulted in the isolation of partial LS01 cDNA sequence with poly(A)+ tail. Subsequently 5′-RACE reaction produced a full-length sequence of LS01. This clone was found to exhibit 86% and above homology at the amino acid level with the deduced amino acid sequences of Lhcb1 of photosystem 11 cDNAs isolated from 9 different monocot and dicot plants (GenBank database). Furthermore, the ORF of LS01 gene also encodes for both transit peptide and mature protein. The transit peptide is required for the transportation of LS01 gene into the chloroplast.
The gene copy number of LS01 was determined by Southern blot analysis. The 3′ end gene-specific probe used in the analysis was able to distinguish LS01 from other members of this gene family. Only one copy of this gene is found in the oil palm genome. In the Northern analysis, expression of LS01 transcript was high and strong in the young and mature green leaves. As for yellowish spear leaves, lower level of expression was observed. However, the expression of LS01 transcript was not detected in the non-photosynthetic tissues such as kernel, mesocarp, germinated seedlings and flower.
The genome walking approach was successfully used for isolating the LS01 promoter. The presence of gene-specific primers in both primary and secondary PCR was able to amplify the genomic clone of interest from a pool of digested and adaptor ligated genomic DNA. In addition, the same approach was also utilized to study the structure of LS01 genomic clone. It was observed that introns were absent from the LS01 genomic sequence.
The strength and specificity of LS01 promoter was confirmed by a transient assay system and transgenic analysis using model plant, Arabidopsis thaliana. In the transient GUS assay, LS01 promoter was cloned into pB122I vector carrying GUS as a reporter gene after removal of 35S CaMV promoter. As for transient GFP assay, LS01 promoter was cloned into pEGFP promoterless vector carrying GFP as a reporter gene. Both of the plasmids DNA were used in the bombardment of oil palm leaf tissues and mesocarp slices as control. Results obtained from the GUS assay and GFP detection confirmed that LS01 promoter was able to drive the expression of the reporter genes only in the leaf tissue. In the Arabidopsis work, the model plant was transformed with Agrobacterium carrying a binary vector harboring the leaf-specific promoter controlling a reporter gene (GUS). Transgenic plant carrying the leaf specific promoter was planted until the third generation in order to obtain a stable integration of the transgenes in the Arabidopsis genome. Results from the GUS staining of the Arabidopsis seedling further confirmed the leaf specificity of LS01 promoter.
Accordingly, it is the primary object of the present invention to provide a promoter sequence of chlorophyll a/b binding protein gene isolated from oil palm, wherein the promoter sequence exhibits leaf-specificity.
It is another object of the present invention to provide a promoter sequence for controlling leaf-specific expression of foreign genes encoding protein.
It is another object of the present invention to use the complete or partial sequence of LS01 cDNA or promoter for isolation of promoter or regulatory sequence.
It is yet another object of the present invention to provide a recombinant DNA construct containing LS01 promoter for transforming plant cells, plant tissues or parts of plants.
It is yet another object of the present invention to provide transgenic plants resulting from recombinant DNA constructs, to produce high value-added products, monoclonal antibodies, vaccine and other useful industrial or pharmaceutical products.
These and other objects of the present invention are accomplished by providing,
An isolated nucleic acid comprising a regulatory nucleic acid sequence that is at least 50% identical to the sequence set forth in SEQ ID NO: 1 or a complement thereof after optimal alignment.
and
An isolated nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence that has greater than 70% similarity to SEQ ID NO: 2 after optimal alignment.
and
A nucleic acid construct, comprising a nucleic acid as claimed in the present invention, wherein the nucleic acid is operably linked to a recombined nucleic acid.
and
A vector comprising the nucleic acid construct as claimed in the present invention.
and
The nucleic acid construct as claimed in this invention, wherein the recombined (nucleic acid) encodes a protein that impart insect resistance, production of bioplastic, production of nutraceutical products, production of pharmaceutical macromolecules including therapeutic and diagnostic protein, antibodies and vaccines or result in an increase in photosynthetic rate of plant, or result in changes of plant shade.
and
A cell comprising the nucleic acid construct as claimed in the present invention.
and
A transgenic plant comprising the nucleic acid construct as claimed in the present invention.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the products of RT-PCR using primer CAB(F) and CAB(R). Lane M is the DNA Ladder Mix Marker. Lane 1 and 2 are the 500 bp products amplified from the pool of expressed gene in oil palm leaves.
FIG. 2 provides the nucleotide (SEQ ID NO: 39) and deduced amino acid (SEQ ID NO:4) sequences of pRTLS01. The amino acids are shown in single letter codes. The sequence is part of Lhcb gene coding region.
FIG. 3 shows the results of digested phagemids on 1.0% agarose gel. The phagemids were obtained from in vivo excision of putative clones from secondary library screening. Lane M is the DNA Ladder Mix Marker. Lanes 1, 3, 5 and 7 are the undigested phagemids. Lanes 2, 4, 6 and 8 are the phagemids digested with EcoR I and Xho 1.
FIG. 4 provides the nucleotide (SEQ ID NO:5) and deduced amino acid (SEQ ID NO:6) sequences of pLS01 which was obtained from the screening of oil palm leaf cDNA library. The derived amino acids are presented in single letter codes. The consensus sequences for polyadenylation signal are underlined. The stop codon (TGA) is denoted by asterisk (*).
FIG. 5 shows the alignment between deduced amino acid sequences of pRTLS01 (SEQ ID NO:4) and pLS01 (SEQ ID NO:6). Identical amino acids in both sequences are presented by asterisk (*). A total of 96% homology was observed.
FIG. 6 shows the products of SMART RACE that was amplified from the 5′- RACE-Ready cDNA using gene-specific primer, LS 10. Lane M is the DNA Ladder Mix Marker. Lanes 1, 2 and 3 are the 1.0 kb products of SMART RACE.
FIG. 7 provides the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:2) sequences of LS01 complete cDNA sequence. The derived amino acids are presented in single letter codes. The putative transcription start site is bold and italic. The translation start site is indicated in boldface. The transit peptide is underlined. The position marked by brackets denotes the first predicted amino acid of the mature protein. The consensus sequences for polyadenylation signals are underlined and italic. The stop codon (TGA) is denoted by asterisk (*).
FIG. 8 provides the comparison of the deduced amino acid sequence of LS01 (SEQ ID NO:2) with Lhcb I amino acid sequences from other plants. The conserved first predicted amino acid of the mature protein was underlined. Dots have been introduced to optimize alignment. Asterisk (*) represent identical amino acids. The GenBank accession number of the sequences are as follows: duckweed (L. gibba AAA33396; SEQ ID NO:29), cotton (G. hirsutum AAA18529, SEQ ID NO:30), potato (S. tuberosum AAA80589, SEQ ID NO:31), tobacco (N. sylvestris BAA25388, SEQ ID NO:32), tomato (L. esculentum AAA34137, P07370, SEQ ID NO:33), soya bean (G. max AAA50172, SEQ ID NO:34), wheat (T. aestivum P04784, SEQ ID NO:35), rice (O. sativa P12330, SEQ ID NO:36) and maize (Z. mays P06671, SEQ ID NO:37).
FIG. 9 provides the results of Northern blot analysis using two different PCR produced probes, LS1 and LS2 containing the entire sequence of LS01 (9a) and 3′- UTR of LS01 (9b), respectively. Total RNA (5 μg/lane) was size fractionated on 1.2% agarose gel and transferred to nylon membrane prior to hybridization with 32P-labeled LS1 and LS2 probes. M, S, Y, F and GS represent total RNA isolated from mature leaves, spear leaves, young leaves, flower and germinated seedlings. The alphabet ‘w’ represents week after anthesis.
An ethidium bromide stained gel (9c) was included to show the equal loading of total RNA from various oil palm tissues.
FIG. 10 gives the results of Southern analysis for determination of LS01 gene copy number in the oil palm genome. A total of 10 μg genomic DNA from oil palm leaves was digested with Hind III (Lane H) and Xba I (Lane X) prior to size fractionation on 1.0% agarose gel (10a). The digested DNA was transferred to nylon membrane and hybridized with 32P-labeled probes prepared using the entire sequence of LS01 (10b) and 3′-UTR of LS01 (10c).
FIG. 11a shows the amplified PCR products obtained from primary PCR of GenomeWalker libraries using primers LS14 and AP1. Lane M is the DNA Ladder Mix Marker. Lanes 1, 2, and 3 are the products amplified from Dra I, EcoR V, and Pvu II GenomeWalker libraries, respectively.
FIG. 11b shows the amplified PCR products obtained from secondary PCR of Dra I GenomeWalker library using primers LS12 and AP2. Lane M is the DNA Ladder Mix Marker. Lanes 1, 2, 3, 4, 5 and 6 are the products amplified from Dra I GenomeWalker library.
FIG. 12 provides the nucleotide sequence of the oil palm LS01 promoter, pGWLS01 (SEQ ID NO:1). Putative transcription start site is shown in italics. Asterisk (*) represent the overlapping sequences with 5′-UTR of LS01 gene. Several putative cis-acting elements were identified and underlined. These consist of initiator element (Inr), I-box, GATA box, CCAAT box, G-box, wound responsive element (WUN), abscisic acid responsive element (ABA) and heat-shock responsive element (HSE).
FIG. 13a shows the amplified primary PCR products from Dra 1, EcoR V, Pvu 11 and Stu I GenomeWalker libraries, respectively using primers LS17 and API. Lane M is the DNA Ladder Mix Marker. Lanes 1, 2, 3 and 4 are the products amplified from Dra 1, EcoR V, Pvu 11 and Stu I GenomeWalker libraries, respectively.
FIG. 13b shows the amplified secondary PCR products from Pvu 11 GenomeWalker library using primers LS 18 and AP2. Lane M is the DNA Ladder Mix Marker. Lanes 1 and 2 are the products amplified from Pvu 11 GenomeWalker library.
FIG. 14 provides the alignment result of genomic clone, pGWLS1718 (SEQ ID NO:7) and cDNA clone, LS01 (SEQ ID NO:3). Asterisk (*) represent the conserved nucleotides in both sequences. The translation start site (ATG) is bold and italic. Transcription start site is bold and underlined.
FIG. 15 shows the results for restriction enzymes analysis of plasmid pLS01 GUS and pLS01GFP using Sma I and Hind 111. Lane M is the DNA Ladder Mix Marker. Lanes 1 and 3 are the undigested plasmid of pLS01 GUS and pLS01GFP, respectively. Lanes 2 and 4 are the digested plasmid of pLS01 GUS and pLS01GFP, respectively.
FIG. 16 shows histochemical localization of GUS expression in oil palm leaves bombarded with pLS01 GUS plasmid (16a and 16b) and without plasmid (16c). The presence of blue spots were observed in the leaves bombarded with pLS01GUS (16a and 16b) but not in the negative control (16c).
FIG. 17 provides the transient GFP expression in leaf and mesocarp tissues bombarded with different promoter-reporter gene constructs. Leaf discs and mesocarp slices were bombarded with three different plasmids: promoterless pEGFP (negative control), pEGFP driven by LS01 promoter (designated pLS01GFP) and pEGFP driven by constitutive 35S cauliflower mosaic virus promoter (p35SGFP). Expression of GFP was observed in the leaf tissues bombarded with pLS01GFP and p35SGFP. However expression of GFP was not detected in the mesocarp slices bombarded with pLS01GFP.
FIG. 18 shows restriction enzyme analysis of pB1I01LS01 plasmid using Sma I and Hind 111. Lane M I and M2 is DNA ladder mix and λ Hind III marker, respectively. Lanes I and 3 are undigested pBI101LS01 plasmid. Lanes 2 and 4 are pBI101LS01 plasmid digested with Sma I and Hind 111.
FIG. 19 provides PCR analysis of pBI101LS01 plasmid which was isolated from Agrobacterium tumefaciens C58 using LS01 promoter specific primers, LS221c and LS221d. Lane M is the DNA ladder mix marker. Lane I is the undigested pBI101LS01 plasmid which was isolated from Agrobacterium selected on LB agar plate containing 50 μg/ml kanamycin and rifampycin. Lanes 2, 3 and 4 are the PCR products amplified from plasmid in lane 1. Lane 5 is the undigested pBI101LS01 plasmid which was isolated from Agrobacterium selected on LB agar plate containing 50 μg/ml kanamycin. Lanes 6, 7 and 8 are the PCR products amplified from plasmid in lane 5. Lane 9 is the water negative control.
FIG. 20 shows PCR analysis of pBI101LS01 plasmid which was isolated from Agrobacterium tumefaciens C58 using promoter specific primer, LS221c and GUS gene specific primer, GUS-lower. Lane M is the DNA ladder mix marker. Lane 1 is the amplified PCR product for pBI101LS01 plasmid which was obtained from Agrobacterium selected on LB agar plate containing 50 μg/ml kanamycin and rifampycin. Lane 2 is the water negative control.
FIG. 21 shows the germination of seeds on kanamycin selection medium to identify successfully transformed Arabidopsis thaliana progeny. Transformant is resistant towards kanamycin and will grow into green and healthy seedlings (21 a). However, non-transformant will remain as two yellowish leaves seedlings even though it was maintained on the selection medium for 1 month (21 b).
FIG. 22 shows the PCR amplification of partial GUS gene to confirm the presence of pBI101LS01 construct in the putative Arabidopsis thaliana transformant. Genomic DNA extracted from the leaves of transformant was amplified with primers specific for GUS gene (GUS forward, GUS3FOR and GUS reverse, GUS2REV). Lane M is the DNA ladder mix marker. A fragment of 348 bp was successfully amplified in Lane 1 and indirectly confirmed the presence of leaf-specific promoter in the transformant. GUS gene was not detected in wild type genomic DNA (Lane 2) and Lane 3 is the water negative control.
FIG. 23 provides the histochemical GUS staining of 22 days Arabidopsis seedlings from plants transformed with gene construct containing oil palm leaf-specific promoter and 35S CaMV constitutive promoter. Wild type plant was used as negative control. In the seedlings transformed with leaf-specific promoter, blue staining was observed only in the leaf (23a). Blue staining was detected in all the tissues (leaf, stem and root) for plant transformed with constitutive promoter (23b). Blue staining was not detected in the wild type plant (23c).



BRIEF DESCRIPTION OF THE TABLES












Sequence
Sequence
Description


ID No.
identity
of sequence

















1
pGWLS01
Oil palm chlorophyll a/b binding protein




(LS01) promoter sequence


2
LS01
Oil palm chlorophyll a/b binding protein




amino acid sequence


3
LS01
Oil palm chlorophyll a/b binding protein




gene complete ORF sequence


4
pRTLS01
Partial LS01 isolated from RT-PCR, amino




acid sequence


5
pLS01
Partial LS01 isolated from cDNA library,




nucleotide sequence


6
pLS01
Partial LS01 isolated from cDNA library,




amino acid sequence


7
pGWLS1718
Genomic sequence obtained from PCR using




primers LS17 and LS18


8
CAB(F)
Forward degenerate primer for amplification




of partial LS01 using RT-PCR


9
CAB(R)
Reverse degenerate primer for amplification




of partial LS01 using RT-PCR


10
LS1
Entire sequence of LS01 cDNA fragment




amplified for Northern blot analysis


11
LS2
3′-UTR of LS01 cDNA fragment amplified




for Northern blot analysis


12
LS10
Antisense primer for amplification of LS1




and LS2


13
LS11
Sense primer for amplification of LS2


14
LS12
Nested gene-specific primer for amplification




of pGWLS01


15
LS14
Gene-specific primer for amplification of




pGWLS01


16
LS15
Sense primer for amplification of LS1


17
LS17
Gene-specific primer for amplification of




pGWLS1718


18
LS18
Nested gene-specific primer for amplification




of pGWLS1718


19
AP1
Adaptor-specific primer for amplification




of pGWLS01 and pGWLS1718


20
AP2
Nested adaptor-specific primer for amplifi-




cation of pGWLS01 and pGWLS1718


21
LS221c
Sense primer for amplification of leaf-




specific promoter fragment from plasmid




pGWLS01


22
LS221d
Antisense primer for amplification of leaf-




specific promoter fragment from plasmid




pGWLS01


23
EGFPN
Primer for sequencing of plasmid pLS01GFP


24
M13F
Forward primer for sequencing of plasmid




pLS01GUS


25
M13R
Reverse primer for sequencing of plasmid




pLS01GUS


26
GUS-lower
Primer for detection of gus gene in DNA




construct pBI101LS01


27
GUSFOR
Forward primer for detection of gus gene




in genomic DNA of transgenic plant


28
GUSREV
Reverse primer for detection of gus gene




in genomic DNA of transgenic plant


29
Solanum
Solanum tuberosum Lhcb I amino acid



tuberosum
sequence


30
Nicotiana
Nicotiana sylvestris Lhcb I amino acid



sylvestris
sequence


31
Lycopersicon
Lycopersicon esculentum Lhcb I amino



esculentum
acid sequence


32
Gossypium
Gossypium hirsutum Lhcb I amino acid



hirsutum
sequence


33
Glycine max
Glycine max Lhcb I amino acid sequence


34
Lemna gibba
Lemna gibba Lhcb I amino acid sequence


35
Zea mays
Zea mays Lhcb I amino acid sequence


36
Oryza
Oryza sativa Lhcb I amino acid sequence



sativa


37
Triticum
Triticum aestivum Lhcb I amino acid



aestivum
sequence


38
WUN
Wound-responsive element


39
pRTLS01
Partial LS01 isolated from RT-PCR,




nucleotide sequence









DETAILED DESCRIPTION OF THE INVENTION
Sense degenerate primer, CAB(F) and antisense degenerate primer, CAB(R) which were derived from the conserved region of light harvesting chlorophyll a/b binding protein (Lhcb) gene from three different monocotyledonous plants consist of maize, rice and grain were used to amplify the related gene from oil palm cDNA pool. Incorporation of these primers in the PCR reaction resulted in the isolation of 500 bp Lhcb gene from the single stranded leaf cDNA pool derived from reverse transcription of leaf MRNA (FIG. 1). The nucleotide and deduced amino acid sequence of this clone was designated pRTLS01 (FIG. 2). BLASTX analysis using GenBank non-redundant database showed that pRTLS01 has 85% and 84% homology with deduced amino acid sequences from maize and rice, respectively. These homologies were expected as the degenerate primers were designed based on the maize and rice sequences. Sequence of pRTLS01 was found to code for open reading frame (ORF) of Lhcb gene.
Primary screening of leaf cDNA library using radioactive labeled pRTLS01 resulted in the detection of 61 putative oil palm Lhcb clones. After secondary screening, the number of putative clone was reduced to 30. In vivo excision and restriction enzyme analysis of the isolated recombinant phagemids prior to sequencing confirmed that all the clones have the same insert size of 350 bp (FIG. 3). Based on the nucleotide and deduced amino acid sequences, this clone designated pLS01 consists of 224 bp ORF, 89 bp 3′-untranslated region (3′-UTR) and poly(A)+ tail (FIG. 4). A stop codon(TGA) was observed at position 225 bp. Furthermore, two imperfect consensus signals for polyadenylation, AATAAA and TGTGTTTT were also found at 14 bp and 48 bp, respectively downstream from the TGA stop codon. Both of the imperfect sequences motif were also observed in the 3′-UTR of Scots pine Lhcb cDNA (Jansson & Gustafsson 1990). The ORF of pLS01 also showed a very high homology, 96% with part of the ORF from pRTLS01 (FIG. 5).
5′-RACE approach was carried out to obtain the full-length cDNA sequence and to determine the transcription start site of oil palm Lhcb gene. Based on the sequence information of pLS01 an antisense gene-specific primer, LS 10 located prior to the poly(A)+ tail of this clone was designed. The presence of primer LS10 and adaptor primer from the kit in the PCR reaction of 5′-RACE-Ready cDNA had resulted in the amplification of 1.0 kb distinct band (FIG. 6). The nucleotide and deduced amino acid sequences of this clone were designated LS01 (FIG. 7). Analysis on both of the sequences revealed that LS01 represent the full-length clone of Lhcb gene in oil palm. The adenine at the 5′ end of the LS01 sequence is predicted to be the putative transcription start site of this gene. This clone contains a 5′-untranslated region of 78 bp, an ORF of 795 bp and a 3′-untranslated region of 89 bp. The ORF of this clone was found to encode for 265 amino acid protein. A total of 33 amino acids make up the transit peptide and another 232 make up the mature protein. As Lhcb gene is a nuclear encoded gene, transit peptide is required for the transportation of this gene into the chloroplast (Mullet 1993). Transit peptide was also found in the Lhcb gene isolated from other plant species such as maize, tobacco, rice and tomato (Demmin et al. 1989).
Table 1 shows the results obtained from BLASTX identity search for LS01 clone using
GenBank database. This clone exhibited 86% and above homology with the deduced amino acid sequence of photosystem 11 isolated from 9 different monocot and dicot plants. Alignment of LS01 deduced amino acid sequence with the sequences from other plant species in FIG. 8 showed that the region coding for mature protein in highly conserved as compared to the region of transit peptide. Furthermore, a conserved amino acid motif for the start of mature protein, MRK was also observed in all the plants.










TABLE 1






GenBank
Score
Percentage of


Organisms
Identification No.
Value
Identity (%)







Solanum tuberosum
AAA80589
509
92


Nicotiana sylvestris
BAA25388
508
93


Lycopersicon esculentum
AAA34147
508
92


Gossypium hirsutum
AAA18529
506
93


Glycine max
AAA50172
499
92


Lemna gibba
AAA33396
496
90


Zea mays
P06671
481
88


Oryza sativa
P12331
475
89


Triticum aestivum
P04784
469
86









In the Lhcb multigene family of photosystem II, different types of Lhcb (Lhcb1, Lhcb2 and Lhcb3) can be identified based on the amino acid characteristic of transit peptide and mature protein of cDNA clone or the presence of intron in the genomic clone (Demmin et al. 1989; Chinn et al. 1995). The oil palm full-length LS01 has the comparable structure and number of amino acid as observed in the Lhcb1 of Glycine max (Stockinger & Walling 1994), Gossypium hirsutum (Anderson et al. 1993) and Solanum tuberosum (Fernandez et al. 1995). According to Buetow et al. (1988), Lhcb1 ORF normally comprises of 31 to 37 amino acids for transit peptide and 231 to 235 amino acids for mature protein. Based on the results of BLASTX identity search and characteristic of amino acid, it is confirmed that oil palm LS01 belongs to the Lhcb1 gene family.
Northern blot analysis was carried out to determine the expression patterns of the oil palm Lhcb gene in various oil palm tissues. Two PCR produced probes LS1 and LS2 containing entire sequence of LS01 and 3′-UTR of LS01 respectively were hybridized with the Northern blots. It was observed that both of the probes hybridized to a single transcript of approximately 1.0 kb (FIGS. 9a and 9b). Furthermore, the expression of LS01 gene was very strong, specific and developmentally regulated in the leaf tissues. High expression of LS01 was detected in the young and mature green leaves. As for yellowish spear leaves, lower level of expression was observed. The expression of LS01 was not detected in the non-photo synthetic tissues such as kernel, mesocarp, germinated seedlings and flower.
Intensity of the signal observed in the nylon membrane hybridized with probe LS 1 is higher than with probe LS2. The differences indicated that the entire sequence of LS01 hybridized with the entire mRNA transcript that contained the conserved coding region of Lhcb gene. Whereas the 3′-UTR probe enable the specific analysis of individual gene because only LS01 transcript was hybridized. These results revealed that beside LS01, other members of the Lhcb gene could be present in the oil palm leaves.
Genomic Southern analysis was performed to determine the gene copy number of the oil palm LS01. The same PCR produced probes for Northern analysis were used to hybridize to the Southern blots containing genomic DNA digested with Hind III (Lane H) and Xba I (Lane X). It was observed in FIG. 10a that the entire sequence of LS01 hybridized to 4 different fragments in both lanes. However, in the membrane hybridized with the 3′-UTR probe, only one fragment was detected in each lane (FIG. 10b). It was found that these bands, 10 kb in Lane H and 2.2 kb in Lane X migrated in the same distance as one of the fragment in the membrane hybridized with the entire sequence of LS01. These results confirmed the presence of only one copy of LS01 gene in the oil palm genome.
In order to obtain the 5′ upstream regulatory sequence of LS01, genome-walking approach was performed using GenomeWalker kit and Advantage Genomic PCR kit from CLONTECH. In the primary PCR of GenomeWalker libraries using a 29-mer gene-specific primer, LS14 and adaptor primer, AP1, genomic fragments with different sizes were amplified from Dra I, EcoR V and Pvu II digested libraries (FIG. 11a). The largest fragment of 1.2 kb was amplified from the Dra I digested library. Whereas PCR products of approximately 800 bp was amplified from both EcoR V and Pvu II libraries. Since the Dra I digested library contained the largest PCR product, it was selected as a template for secondary PCR. The library was diluted 50× and 1 μl was used in the PCR reaction with a 30-mer nested gene-specific primer, LS12, and nested adaptor primer, AP2 (5′ACTATAGGGCACGCGTGGT3′; SEQ ID NO:20). Secondary PCR using Dra I library resulted in the amplification of 1.0 kb PCR product (FIG. 11b). Since the primer LS12 located 228 bp upstream of the primer LS14, smaller size of amplified genomic fragment around 1.0 kb was expected. The fragment was subsequently cloned into TOPO-pCR®II vector and the recombinant clone designated pGWLS01 was sequenced using M13 forward and reverse primer.
Sequencing result of pGWLS01 was shown in FIG. 12. A total of 932 bp nucleotides upstream of the putative transcription start site coding for the promoter region. As for the 58 bp nucleotides downstream of the putative transcription start site, this region overlaps with the 5′-UTR of LS01. At the expected distant 32±7 bp upstream to the transcription start site, no TATA-box consensus sequence was identified (Joshi 1987). However, at position −1 to −7 upstream from the transcription start site, an initiator element (Inr) which is pyrimidine rich, PyTCANTPyPy was observed (Nakamura et al 2002). This finding revealed that the oil palm LS01 promoter is a TATA-less promoter and initiation of basal transcription could be directed by the Inr motif. The absence of TATA boxes in the majority of nuclear encoded photosynthesis genes were reported previously by Nakamura et al (2002). Their studies on 232 promoter sequences strongly suggest that TATA-independent transcription mechanisms play an important role in the regulated expression of photosynthesis nuclear genes.
Furthermore, at the distal region of the promoter, a few interesting cis-acting elements were identified. Light-responsive elements such as GATA, CCAAT, G- and I-box were commonly found in the light-responsive promoter (Arguello-Astorga & Herrera-Estrella 1998). Two separated regions that contain GATA and CCAAT motifs at position −88 and −65 relative to the transcription start site were postulated to be phytochrome-responsiveness. These motifs were also identified in the Lhcb promoter of Lemna gibba and other plants (Kehoe et al. 1994). In the upstream region of the promoter, a putative wound-responsive element (WUN) CAAATTCCAAA (SEQ ID NO:38) nearly identical to the WUN of pathogenesis-related gene, AAATTTCCT in potato was identified at position −464 (Matton et al. 1993). Whereas at position −699 and −878, an abscisic acid-responsive element (Knight et al. 1992) and heat-shock responsive element (Pastuglia et al. 1997) were identified, respectively. The presence of these elements indicated that the expression of LS01 gene could be regulated by environmental cues such as light, mechanical wounding, abscisic acid and heat.
GenomeWalker kit was also used to determine the presence of intron in the LS01 gene. In the primary PCR with primer LS17, an intense genomic fragment was amplified from EcoR V and Pvu II GenomeWalker libraries. No band was observed in Dra I library and only a small fragment about 0.3 kb was amplified from Stu I library (FIG. 13a). Since the fragment from Pvu II library is slightly larger than EcoR V, it was selected for secondary PCR. Further amplification of this template with nested gene-specific primer, LS 18 resulted in the isolation of a 0.9 kb genomic clone (FIG. 13b). Alignment between this sequence, designated pGWLS1718 with full-length LS01 cDNA sequence was shown in FIG. 14. It was found that nucleotides upstream to the transcription start site were similar to the proximal region of LS01 promoter. Whereas a total of 508 bp nucleotides downstream to the transcription start site were identical to the coding region of LS01 gene. No introns sequences were presence in this clone. Such a criterion is only observed in the Lhcb1 gene which is typically lack of introns (Arguello-Astorga & Herrera-Estrella 1998). This result further confirms that oil palm LS01 belongs to the Lhcbl gene family.
Transient expression assay was carried out for analyzing the strength and specificity of oil palm leaf-specific promoter. A 900 bp promoter which was amplified from pGWLS01 using primer LS221c and LS221d has been successfully ligated into promoterless pBI221 and pEGFP vector carrying GUS and GFP, respectively as reporter gene. Restriction analysis of the recombinant plasmids designated pLS01GUS and pLS01 GFP using Sma I and Hind III was shown in FIG. 15. Digested pLS01GUS showed the presence of 5.7 kb pB1221 vector and 900 bp promoter. As for pLS01GFP, fragments for vector and promoter were also observed. The size of the pEGFP vector was 4.2 kb. Analysis of the sequencing results confirmed that LS01 promoter was ligated in the correct orientation in both recombinant plasmids.
In the histochemical localization of GUS expression, GUS enzyme (EC 3.2.1.31) which was encoded by the uidA locus will catalyse the cleavage of the substrate 5-bromo-4-chloro-3 indoyl glucuronide (X-Gluc). Precipitation of blue dye at the site of enzyme activity was obtained through oxidative dimerization of the indoxyl derivative. It was found that the presence of oxidative catalyst such as ferricyanide and ferrocyanide mixture could enhance this dimerization process (Jefferson et al, 1987). In the studies, 2 days of incubation in GUS staining solution resulted in the detection of 60 blue spots in the leaf tissues bombarded with pLS01GUS at 1350 psi helium pressure and 9 cm distance from macrocarrier to target tissues. Moreover, it was also observed in FIG. 16 that some blue spots could be attributed to a single blue cell (16a), but in most of the cases several adjacent cells were also stained (16b). This type of enzyme localization was also detected in the work carried out by Chowdhury et al, (1977). As for the negative control in FIG. 16c, the presence of blue spot was not observed.
Specificity of LS01 promoter was further proven by the result obtained from GFP detection. Bombardment of leaf discs and control mesocarp slices with pLS01GFP showed a very promising result. Using this construct, GFP expression was detected only in the leaf but not in the mesocarp slices (FIG. 17). As for the construct driven by constitutive 35S promoter, green fluorescence spots were detected in both tissues. No expression of GFP was detected in the tissues bombarded with promoterless pEGFP.
For a stable integration of LS01 promoter into the Arabidopsis genome, the first step involved the construction of a suitable recombinant binary vector and cloning in E. coli. Transformation of the ligated product containing the oil palm leaf-specific promoter (LS01) and pBI101 binary vector into E.coli strain DH5α resulted in the isolation of two single bacteria colonies. The recombinant plasmids designated pBI101LS01 were then extracted from these colonies. As shown in FIG. 18, two different sizes of bands which were 12.5 kb vector and 0.9 kb LS01 fragment were observed after the pBI101LS01 plasmid was digested with Sma I and Hind 111. This analysis confirmed that LS01 has been successfully cloned into pBI101. Whereas analysis of the sequencing results showed that LS01 was ligated into pB101 in the correct orientation.
In order to enable the transfer of construct of interest into Arabidopsis, pBI101LS01 plasmid was transformed into the Agrobacterium tumefaciens strain C58 which has often been called ‘nature's genetic engineer’ using electroporation method. After 3 days incubation at 28° C., Agrobacteriumcolonies were observed in the agar plate containing kanamycin. It was found that the actual volt of 2.21 kV and time constant of 5.2 mseconds yielded bigger size colonies. Subsequently, a total of 8 transformants were grown in LB broth containing kanamycin and plasmids were prepared using QIAPREP spin miniprep kit (QIAGEN). On the other hand, glycerol stocks of the same plasmids were also restreaked on LB plate containing rifampycin and kanamycin. Plasmids were also prepared from these transformants. As shown in FIG. 19, transformants obtained from two different antibiotic selection plates showed the presence of 0.9 kb LS10 promoter fragment. This result suggested that kanamycin alone was able to select recombinant Agrobacterium. In order to verify accurately the presence of T-DNA plasmid in the transformants, PCR analysis was repeated using leaf promoter and GUS specific primers. It was observed in FIG. 20 the amplification of 2.7 kb PCR product. The expected size comprised of 1.8 kb GUS gene (Mayer et al., 2001) and 0.9 kb LS01 promoter fragment. Based on these results, the transformed Agrobacterium can be used for transforming Arabidopsis thaliana. 
In planta transformation of Arabidopsis was performed via floral dip method. According to Weigel and Glazebrook (2002), this method was able to give transformant frequency of 0.1 to 1%. In the work carried out by Clough and Bent (1998), they concluded that inflorescence developmental stage and inoculation medium were the most important factors that determine the efficiency of floral dip transformation. Plants with the maximum number of unopened floral buds were the most susceptible stage for transformation. Whereas with the presence of 5% sucrose in the inoculum, a total of 1.62% transformed Arabidopsis was obtained. As for surfactant SILWET L-77, this component greatly enhances the entry of bacteria into relatively inaccessible plant tissues. In this study, plants with many immature floral bud and few opened flowers were chosen for dipping in the inoculation medium containing LB broth, 5% sucrose and 0.05% SILWET L-77. After 8 weeks on the soil, seeds were collected from the T1 plant.
Selection of putative transformant from T1, seeds was carried out on the kanamycin selection medium. After 14 days, transformant was observed in one of the plate. The transformant was a kanamycin resistant seedling that produced green leaves and able to develop into a mature Arabidopsis plant (FIG. 21a). As for non-transformant, the seedling will only have two yellowish leaves and the growth was retarded (FIG. 21b). On the third week, the number of adult leaves of the transformed Arabidopsis has increased from 3 to 5 leaves. At this stage, the seedling was transplanted into soil. Seeds collected from this plant were designated as T2.
Screening of 16 plants from T2 generation resulted in the isolation of one homozygous line (designated as T1P1,/T2P4) which exhibited 100% survivor rate on the kanamycin selection medium. PCR analysis had successfully amplified a 348 bp partial GUS gene from the leaf genomic DNA of this homozygous plant (FIG. 22). This further confirmed the presence of pBI101LS01 construct in the transformed plant. In addition, GUS assay of 22 days seedlings also showed the specific expression in the leaf tissue. GUS staining was observed only in the leaf tissue but not in the stem and root (23a). However for the plant transformed with the gene construct containing 35S constitutive promoter, all the tissues stated above were stained blue (23b). As for wild type plant, no blue staining was obtained (23c).
Based on the results from transient expression assay and stable transformation in the Arabidopsis, it can be concluded that the LS01 promoter was able to drive the expression of transgenes specific to the leaf tissue. Furthermore, successful transformation of this promoter into the Arabidopsis also showed that it can be used in the heterologous plant system.
EXAMPLES
Example 1
Amplification of Lhcb Gene Via Reverse Transcription and Polymerase Chain Reaction (RT-PCR) Approach
First strand cDNA was synthesized from leaf mRNA in 25 μl reaction containing 5 μg leafmRNA, 4 μl 5× first strand buffer, 2 μl 0.1 M DTT, 5 μl 2 mM dNTP and 1 μl SUPERSCRIPT II reverse transcriptase (200 U/μl ) (Gibco BRL Life Technology Inc. N.Y., USA) at 42° C. for 1 hour. The solution was phenol-chloroform extracted, ethanol precipitated and the pellet was dissolved in 25 μl sterile water. The cDNA was subjected to RNA hydrolysis using 12.5 μl 0.15 N NaOH and 1 μl 0.5 M EDTA at 68° C. for 15 min. This was followed by neutralization of the reaction by addition of 12.5 μl Tris-HCI, pH 8.0; 12.5 μl 1 N HCl and 17.3 μl 7.5 M ammonium acetate prior to ethanol precipitation. The cDNA pellet was dissolved in 25 μl sterile water and the concentration of cDNA was determined using ethidium bromide plate.
PCR was carried out with the presence of degenerate primers [CAB(R)-5′CNGGRTCNGCDATRTGRT3′ (SEQ ID NO:9) and CAB(F)-5′GCNGAYCCNGARACNTTY3′ (SEQ ID NO:8)] which were designed based on the conserved region of known chlorophyll a/b binding protein gene from maize, grain and rice. The 50 μl PCR mixture contained 1 μl cDNA (50 ng),5 μl 10× buffer, 1 μl 10 mM dATP, 1 μl 10 mM dCTP, 1 μl 10 mM dGTP, 1 μl 10 mM dTTP, 2 μl 15 μM CAB(R), 2 μl 15 μM CAB(F), 5 μl 25 μM MgCl2, 30.5 μl sterile water and 0 μl Amplitaq DNA polymerase(5 U/μl). The PCR reaction was placed in the Perkin Elmer 9700 thermo cycler with the following conditions: 94° C., 5 min for 1 cycle; followed by 94° C., 1 min; 43° C., 1 min and 72° C., 1 min 30 sec for 40 cycles and finally 72° C., 10 min for 1 cycle. The expected fragment was purified using QIAQUICK Gel Extraction Kit (QIAGEN), cloned into TOPO-pCR®II vector from TOPO-TA Cloning Kit (Invitrogen) before subjected to automated sequencing with ABI 377 PRISM. Analysis of the nucleotide sequences were performed using DNASIS Max version 1.0 prior to database homology search.
Example 2
Screening of Leaf CDNA Library With cDNA Probe Generated From RT-PCR
A total of 200,000 plaques from leaf cDNA library constructed in Uni-ZAP XR vector (Stratagene) were plated based on Sambrook et al (1989) and plaque lift was performed as described by Siti Nor Akmar et al (1995). The plaques lifted membranes were first treated with denaturation buffer (0.5 N NaOH, 1.0 M NaCl) for 10 min, followed by neutralization buffer (0.5 M Tris-HCl, pH 8.0; 1.5 M NaCl) for 5 min and 2×SSC for 5 min prior to optimal crosslinked with UV light.
Prehybridization of the membranes were carried out at 65° C. in 5× Denhardt's solution (1× Denhardt's solution is 0.02% each Ficoll 400, bovine serum albumin and polyvinylpyrrolidone), 5× SSPE (1× SSPE is 0.18 M NaCl, 10 M NaH2PO4, pH 7.5, 1 mM EDTA), 0.5% SDS and 100 μg/ml denatured herring sperm DNA. Hybridization of the membranes was performed using the same hybridization buffer with the presence of 32P-labelled pRTLS01 at 65° C. The probe was labeled with 32P-dCTP using MEGAPRIME DNA Labeling Kit from Amersham Pharmacia Biotech. After overnight hybridization, the membranes were washed at 65° C. with 4× SSPE, 0.1% SDS; followed by 2× SSPE, 0.1% SDS and 0.5× SSPE, 0.1% SDS. These membranes were then exposed to x-ray film for 24 hours at −80° C.
Based on the signal detected on x-ray film, putative plaques were cored out and placed into SM buffer (100 mM NaCl, 10 mM MgSO4.7H20, 0.05 M Tris-HCI and 0.01% gelatin) containing 0.3% chloroform. The phage lysate was subjected to PCR analysis with the presence of primer T7 and gene-specific primers. The PCR conditions were as follows: 95° C., 5 min and 80° C., 45 min for 1 cycle; followed by 95° C., 1 min; 60° C., 1 min and 72° C., 1 min 30 see for 30 cycles and 1 cycle of final extension at 72° C. for 10 min. Amplified products were visualized on 1.2% agarose gel.
Recombinant phage was in vivo excised according to the manufacturer's instruction (Stratagene). The purified pBluescript phagemid was digested with EcoR I and Xho I to confirm the length of cDNA insert.
Example 3
Rapid Amplification of 5′-cDNA Ends (5′-RA CE)
A full-length sequence of chlorophyll a/b binding protein gene was isolated via 5′ RACE using SMART RACE cDNA amplification Kit and Advantage 2 PCR Kit from CLONTECH, Laboratories, Inc., USA. The reaction for first strand cDNA was initiated by incubation of 1 μg total RNA from leaf with 1 μl 5′-CDS primer, 1 μl SMART II A oligo and 2 μl sterile water at 70° C. for 2 min. The reaction was immediately cooled on ice prior to addition of 2 μl 5× first strand buffer, 1 μl 20 mM DTT, 1 μl 10 mM dNTP mix and 1 μl reverse transcription PowerScript. This was followed by incubation at 42° C. for 1.5 hours. The reaction was stopped by addition of 200 μl Tricine-EDTA buffer and incubation at 72° C. for 7 min.
A total of 2.5 μl single stranded 5′-RACE Ready cDNA was added to the PCR mixture containing 34.5 μl PCR grade water, 5 μl 50× Advantage 2 polymerase buffer, 5 μl 10× UPM primer and 1 μl 10 μM gene-specific primer, LS10 (5′TAATGCACACCACGCCAACAATTTCAATTC3′; SEQ ID NO:12) which was designed based on the sequence of pRTLS01. The PCR reaction was carried out using Perkin Elmer9700 thermo cycler with the following conditions: 94° C., 5 sec; 68° C., 10 sec and 72° C., 3 min for 25 cycles. The expected fragment was purified, cloned into TOPO-pCR®II vector from TOPO-TA Cloning Kit (Invitrogen) before subjected to automated sequencing with ABI 377 PRISM. Analysis of the nucleotide sequences were performed using DNASIS Max version 1.0.
Example 4
Northern Blot Analysis
Total RNA was extracted from various tissues of oil palm according to the method of Rochester et al. (1986).
Two different fragments containing complete nucleotide sequence and 3′ untranslated region (3′-UTR) of chlorophyll a/b binding protein gene were used as probes in the Northern and Southern analysis. The complete nucleotide sequence of LS01 was generated through amplification of plasmid LS 10.3 with primer LS 10 (5′TAATGCACACCACGCCAACAATTTCAATTC3′; SEQ ID NO:12) and LS15 (5′GCACCTACCCAACAGCATTTCCATTGG3′; SEQ ID NO:16). Whereas 3′-UTR region was amplified using LS10 and LS11 (5′GCCTGGCAACCTTAATTAATTTGGTGCTTAG3′; SEQ ID NO:13) primer pair. The Expected fragments were purified using QIAQUICK Gel Extraction Kit (QIAGEN) and labeled with 32P-dCTP using MEGAPRIME DNA Labeling Kit from Amersham Pharmacia Biotech.
Northern blot analysis has been carried out according to the method of McMaster & Carmichael (1977) and Kroczek & Siebert (1990). In this study, 5 μg of total RNA was heat denatured at 55° C. for 15 min in 18 μl GFP mixture containing 78% (v/v) deionized formamide, 16% deionized glyoxal and 10 mM sodium phosphate buffer. After heat denaturation, the RNA was cooled immediately on ice prior to electrophoresis on 1.2% agarose gel with 40 mM× TAE, pH 7.2 as electrophoresis buffer. The RNA was transferred to HYBOND-N+ membrane (Amersham Pharmacia Biotech) via a vacuum blotter (60 psi, 6 hours) with the presence of 20×SSC as transfer buffer.
Prehybridization of the membrane was performed at 65° C. for 4 hours in 5×SSC (1×SSC is 0.15 M NaCl, 15 mM trisodium citrate), 5× Denhardt's (1× Denhardt's is and 0.02% each Ficoll 400, bovine serum albumin and polyvinylpyrrolidone), 0.5% SDS 100 μg/ml denatured herring sperm DNA. This was followed by overnight hybridization of the membrane with 32P-labeled probe at 65° C. Washing of the membrane was performed with 4×SSC/0.1% SDS at 65° C. for 15 min, followed by 2×SSC/0.1% SDS at 65° C. for 15 min. Exposure to x-ray film was carried out at −80° C. for 48 hours.
Example 5
Southern Blot Analysis
Genomic DNA was extracted from oil palm spear leaves according to the method of Doyle & Doyle (1990).
A total of 20 μg genomic DNA was digested with Hind III and Xba I. The digested products were size fractionated on 1.0% agarose gel at 100 v for 5 hours in 1× TAE, pH 7.9. This was followed by immobilization of the DNA onto nylon membrane via vacuum blotting of the gel at 60 psi for 1 hour with the presence of 0.4 N NaOH as transfer buffer. At the end of the process, the membrane was rinsed with 2×SSC prior to UV-crosslinking. Hybridization and washing of the blot was performed as stated above for Northern blot analysis.
Example 6
Promoter Isolation
Leaf-specific promoter was isolated following the standard protocol stated in the manual of Universal GenomeWalker Kit and Advantage Genomic PCR Kit from CLONTECH Laboratories, Inc. Two antisense gene-specific primers, designated LS14 (5′GTGTCCCACCCATAGTCACCGGGGAATTC3′; SEQ ID NO:15) and LS12 (5′GATGATGCCTTGGAGATGGGAGCGGTGATC3′; SEQ ID NO:14) were designed based on the 5′-terminal of the coding region of LS01 and within 5′-UTR of LS01, respectively. A total of four GenomeWalker libraries were obtained through digestion of 2.5 μg leaf genomic DNA with Dra 1, EcoR V, Pvu 11 and Stu I prior to ligation with the GenomeWalker adaptor. An aliquot of 12 μl of these libraries were used in the adaptor primer, AP2 (5′ACTATAGGGCACGCGTGGT3′; SEQ ID NO:20), supplied in the kit. PCR conditions were carried out as primary PCR reaction with the presence of antisense gene-specific primer, LS 12, and adaptor primer, AP1 (5′GTAATACGACTCACTATAGGGC3′; SEQ ID NO:19) provided with the kit. This was followed by secondary PCR of 50× diluted primary library with antisense nested gene specific primer, LS 14 and recommended in the GenomeWalker Kit manual using Perkin Elmer 9700 thermo cycler. The expected band was purified from agarose gel using QIAquick
Gel Extraction Kit (QIAgen), cloned into TOPO-pCR®II vector from TOPO-TA Cloning Kit (Invitrogen) prior to sequencing using M13 reverse and forward primers.
Genome walking approach was also used to study the structure of LS01 gene. Primary PCR of the GenomeWalker libraries was carried out with the presence of antisense gene-specific primer, LS17 (5′CGAAGTTGGTGGCGTAGGCCCAAGCATTG3′; SEQ ID NO:17) from 3′-terminal of the coding region of pLS01 and primer AP 1. Followed by secondary PCR with antisense nested gene specific primer, LS 18 (5′CTCTGAGCATGGATCAAGCTCGGGTTGCC3′; SEQ ID NO:18) from 5′-terminal of the coding region of pLS01 and primer AP2. The expected band was purified, cloned and sequenced as above.
Example 7
Cloning of the Leaf-specific Promoter into pBI221 and pEGFP Vector
Leaf specific promoter (922 bp) was amplified from plasmid pGWLS01 using sense and antisense primer. The sense primer, LS221c (5′CCCAAGCTTCCATATCTGGCTCG3′; SEQ ID NO:21) was introduced with Hind III site at the 5′ end. As for antisense primer, LS221d (5′TCCCCCGGGCAATGGAAATGCTG3′; SEQ ID NO:22), 5′ end was introduced with Sma I site. These primers, 2 μl of 15 μM stock, were used to amplify the promoter in 50 μl PCR reaction contained 4 μl dNTP (10 mM each), 250 ng plasmid pGWLS01, 5 μl 10× buffer, 5 μl 25 mM MgCl2 and 2.5 U AMPLITAQ DNA Polymerase from Perkin Elmer. PCR conditions were as follows: 95° C., 5 min for 1 cycle; followed by 95° C., 1 min; 56° C., 1 min and 72° C., 1 min 30 sec for 30 cycles and 1 cycle of final extension at 72° C. for 10 min.The PCR product was purified from primers, nucleotides, polymerases and salts using QIAQUICK PCR Purification column from QIAGEN. Plasmid pB1221 carrying GUS as reporter gene and promoterless plasmid, pEGFP carrying GFP as reporter gene were prepared using QIAPREP Miniprep kit from QIAGEN. Fragment of LS01 promoter, plasmid pB1221 and pEGFP were digested first with Sma I (Fermentes) at 30° C. for 6 hours. Digestion with second restriction enzyme, Hind III (Fermentes) was performed at 37° C. for 16 hours. The digested products were analyzed on 1.0% agarose gel and expected fragments were purified from the agarose gel using QIAQUICK Gel Extraction Kit from QIAGEN.
Digested LS01 promoter and promoterless pB1221 at a molar ratio of 4:1 were incubated at 50° C. for 5 min. After immediate cooling, the vector and insert mixture were added into ligation mixture containing 1.5 μl 10× ligase buffer and 1.5 μl T4 DNA ligase (1 U/μl) prior to overnight incubation at 16° C. Ten microlitres of the ligation mixture were used for transformation with competent cell of E.coli DH5α as described by Siti Nor Akmar Abdullah (1999). Blue/white selection of recombinant clones were carried out on LB plate containing 40 μl of 20 mg/ml 5-bromo-4-chloro3-indolyl-β-D-galactopyranoside (X-gal) and 40 μl of 20 mg/ml isopropyl β-D-thiogalactopyranoside (IPTG). Plasmid of the recombinant clone, designated pLS01GUS was prepared using QIAPREP Spin Miniprep Kit (QIAGEN) and digestion with restriction enzymes Sma I and Hind III was performed to confirm the insert size. Lastly, the plasmid was sequenced using M13F (5′GTAAAACGACGGCCAG3′; SEQ ID NO:24) and M13R (5′CAGGAAACAGCTATGAC3′; SEQ ID NO:25) primers. Cloning of the digested LS01 promoter into promoterless pEGFP was also carried out as above and the recombinant clone, pLS01 GFP was sequenced using EGFPN primer (5′CGTCGCCGTCCAGCTCGACCAG3′; SEQ ID NO:23).
Example 8
Promoter Analysis Via Histochemical GUS Assay and GFP Detection
Preparation of Tissue Slices
Oil palm green leaves were collected from seedling palm in MPOB nursery. The tissues were soaked in RBS for 15 min before subjecting to surface sterilization using 20% of CLOROX for 15 min in the laminar flow. The leaves were rinsed twice with sterile water, cut into segments of 1.0 cm2 and were flattened on Murashige and Skoog medium (Duchefa, Biochemicals Plant Cell and Tissue Culture, Haarlem, Netherlands) with the lower epidermis facing upward. Leaf discs were kept at 28° C. for 24 hours and were illuminated before and after bombardment with chimeric gene construct.
Preparation of Gold for Particle Bombardment
A total of 1 ml absolute ethanol was added to 0.06 g of 1.0 micron gold particles. The mixture was mixed by vortexing at high speed. After centrifugation at 10,000 g for 1 min, the supernatant was removed from the gold pellet. These sterilization steps were repeated for 3 times. At the final sterilization, 1 ml sterile water was added to the gold pellet. Sonication of the gold pellet was repeated 3 times before the gold was resuspended in 1 ml sterile water. The gold can be stored at −20° C. for up to 6 months.
Precipitation of DNA Onto Gold Microprojectiles
An approximately 10 μg of plasmid pLS01GUS was added to 2 μg of gold particles in a microcentrufuge tube. While vortexing, 100 μl 2.5 M calcium and 40 μl 0.1 M spermidine was added. The mixture was vortexed for 3 min prior to centrifugation at 10,000 g for 1 min. The supernatant was removed and the microcarrier was resuspended in 65 μl absolute ethanol. The DNA-coated microcarrier can be kept at −20° C. until used.
Microprojectile Bombardment
Leaf discs were bombarded with gene construct using the BioRad (Hercules, Calif., USA) PDS-1000/Helium-driven Particle Delivery System. Before bombardment, the machine chamber, rupture disc, stopping screen, macrocarrier, macrocarrier holder and red caplugs were sterilized with absolute ethanol. An aliquot of 8 μl DNA-coated microcarrier was loaded onto the center of the macrocarrier and air dried for 10 minutes. The leaf discs were bombarded using the following conditions: 1350 psi helium pressure with 9 cm distance from macrocarrier to target tissues. As for mesocarp slices, bombardment was performed at 1550 psi with 9 cm distance from macrocarrier to target tissues. Leaf discs bombarded without plasmid DNA were used as negative control in GUS assay. Whereas in GFP, leaf discs and mesocarp slices bombarded with promoterless pEGFP were used as negative control. After bombardment, the leaf tissues were incubated for 24 hours at 28° C. in the light prior to GUS assay and GFP detection.
Histochemical GUS Assay
GUS activity was measured histochemically following the method described by Jefferson et al. (1987). The leaf tissues were incubated in filter-sterilized GUS staining buffer (0.1 M sodium phosphate buffer, pH 7.0; 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg/ml 5-bromo-4-chloro-3-indoyl glucuronide/X-Gluc, 0.2% Triton X-100 and 10 mM EDTA) for up to 2 days at 37° C. in the dark. After staining, the tissues were fixed in fixative mix (10% formaldehyde, 50% ethanol and 5% glacial acetic acid) and the leaves chlorophyll was removed using 80% ethanol. The presence of blue spots were observed using LEICA QF550W System.
Detection of Green Fluorescent Protein (GFP)
Microscopic detection of GFP was carried out using LEICA MZ 12.5 microscope with blue light. Image of the GFP was captured using Image Manager 50 computer software.
Example 9
Promoter Analysis Using Arabidopsis thialiana (Model Plant System)
Preparation of PB1101LS01 Construct
Fragment of leaf-specific promoter from plasmid pGWLS01 and promoterless binary vector pB 101 (at a molar ratio of 13:1) which have been digested with Sma I and Hind III were heat treated at 55° C. for 5 min. This was followed by immediate cooling and addition of ligation Mixture containing 1.5 μl 10× ligase buffer and 1.5 μl T4 DNA ligase (1 U/μl) prior to overnight incubation at 16° C. Fifty microlitres of the ligation mixture were then used to transform competent cell E.coli DH5α and selection of recombinant clones were carried out on Luria-Bertani (LB) plate containing 50 μg/ml kanamycin. Plasmid of the recombinant clone, designated pBI101LS01 was prepared using QIAPREP spin miniprep kit (QIAGEN) and digestion with restriction enzymes Sma I and Hind III was performed to confirm the insert size. Lastly, the plasmid was sequenced using M 13F and M13R primers.
Electroporation
A total of 100 ng binary plasmid pBI101LS01 was mixed on ice with 100 μl competent cell of Agrobacterium tumefaciens strain C58 in a prechilled Gene Pulser Cuvette (0.2 cm electrode gap). The mixture was subjected to electroporation using Electroporator Gene Pulser®II (Bio-Rad) with the following conditions: capacitance: 1.0 μF, voltage: 2.2 kV and time constant: 5 to 10 mseconds. Immediately after electroporation, 1 ml of LB broth was added to the cuvette prior to incubation at 28° C. for 4 hours. The culture was incubated without shaking for the first 2 hours and continued by gentle shaking at 150 rpm for the following 2 hours. After incubation, the cells were collected by centrifuging briefly at 4000 rpm for 1 min. The pellet was resuspended in 40 μl of LB broth and the cells were plated on LB plate containing 50 μg/ml kanamycin. The plate was incubated for 3 days at 28° C. and PCR was performed using primers specific for leaf promoter and GUS gene (GUS-lower, 5′CATTGTTTGCCTCCCTGCTGCGGTT3′; SEQ ID NO:26) to verify the presence of the recombinant binary vector, pB101LS01 in the Agrobacterium. 
Growing of the Arabidopsis Plant
Seeds from Arabidopsis thaliana ecotype Columbia were germinated and grown to flowering stage in a flower pots filled with a soil mixture of two-thirds Steven Dutch potting mix and one-third vermiculite in a growth chamber. The growth conditions were 16 hours lights (80-100 μmol/m2) at 22° C. 8 h dark at 20° C., with a relative humidity of 75%. In order to obtain more floral bud per plant for dipping, primary inflorescences were clipped to encourage the emergence of secondary bolts.
In Planta Transformation via Floral Din
A single colony of Agrobacterium tumefaciens strain C58 harboring the binary plasmid pBI101LS01 was grown overnight at 28° C. with shaking (220 rpm) in LB broth containing 50 μg/ml kanamycin. The overnight cultures were diluted to 1:10 using LB broth and grown for approximately 8 hours to obtain OD600 of 0.6-0.8. Cells were harvested by centrifugation at 5000 rpm for 15 min. The pellet which was pink in color was then resuspended in 5% (w/v) sucrose. The culture was transferred to a square container and SILWET L-77 was added to 0.05% (v/v). Plants were inverted into this suspension and dipped for 10 seconds. Plants were then removed and the flower pots were enclosed with plastic bags. Top of the plastic bags were closed with paper clip for 2 days to maintain high humidity. After 48 hours, the paper clip was removed and the plants were grown under normal growing conditions. The plant which was designated as T1 were grown to maturity and harvested when the siliques were brown and dry.
Screening and Selection of Putative Transformant
Harvesting of Transgenic Seeds
Seven weeks after floral dip, putative transgenic seeds were harvested by gentle pulling of Dry siliques through fingers over a piece of clean paper. The seeds were sieved to remove the pod materials. Clean seeds were stored in the EPPENDORF tube and kept at 22° C. in the dessicator.
Sterilization of Transgenic Seeds
The seeds were suspended in sterile water containing 1% TWEEN 20 (v/v) in an EPPENDORF tube. The seeds were vortexed, centrifuged and rinsed with sterile water 3 times prior to soaking in 25% Clorox for 20 minutes. Clorox was then removed by washing the seeds with sterile water 3 times. Lastly the seeds were subjected to surface sterilization with 70% ethanol for 1 minute. The seeds were rinsed again with sterile water before it can be used for selection.
Selection for Homozygous Line Carrying Leaf-Specific Promoter
Sterilized seeds were plated on Murashige and Skoog medium supplemented with 50 μg/ml kanamycin. The selection plates were cold-treated at 4° C. in the dark for two days to promote the uniform germination of seeds. The plates were then transferred to 22° C. under continuous fluorescent light illumination. After about 3 weeks, 16 kanamycin-resistant seedlings were transplanted into soil. These T2 generation plants were grown to maturity and individually harvested. Seed sterilization and screening were repeated for these 16 plants. Only those T3 generation seedlings that shown 95% and above survivor rate on the kanamycin selection plate were considered as homozygous line. These plants were then transplanted into soil for seeds collection.
PCR Analysis of Transformant
Genomic DNA was extracted from Arabidopsis leaves carrying leaf-specific promoter according to the method described by Doyle and Doyle (1990). PCR amplification was performed using primers specific for the GUS gene which are GUS3FOR (5′TGACGCATGTCGCGCAAGAC3′; SEQ ID NO:27) and GUS2REV (5′ATCCTTTCGCACGTAAGTCC3′; SEQ ID NO:28). In this study, genomic DNA from wild type Arabidopsis was taken as negative control.
Histochemical GUS Assay of Transformant
GUS activity was measured histochemically following the method described by Jefferson et al. (1987). The whole plant of Arabidopsis seedling (22 days) carrying leaf-specific promoter were incubated in filter-sterilized GUS staining buffer (0.1 M sodium phosphate buffer, pH7.0; 1 mg/ml 5-bromo-4-chloro-3-indoyl-glucuronide/X-gluc and 0.2% Triton X-100) overnight at 37° C. in the dark. After staining, chlorophyll was removed from the plant using 70% ethanol. The presence of blue precipitate were observed using NIKON SMZ800 Stereomicroscope. Blue deposits due to GUS activity in the transformant was compared with Arabidopsis carrying 35S constitutive promoter and also wild type plant.
REFERENCES


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While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications to the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of this invention.

## Claims

1. An isolated nucleic acid comprising the sequence of SEQ ID NO: 1.

2. An isolated nucleic acid effective to regulate expression of an operably linked polynucleotide, wherein the nucleic acid comprises a fragment of the nucleic acid of SEQ ID NO:1 with promoter activity, and wherein the fragment comprises the following elements depicted in FIG. 12: GATA I box, GATA II box, CCAAT box, and the G-box.

3. A nucleic acid construct comprising the isolated nucleic acid of claim 1 operably linked to a recombinant nucleic acid, wherein the nucleic acid construct when introduced into leaf tissues and/or photosynthetic tissues, stimulates expression of the gene recombinant nucleic acid in the leaf tissues and/or photosynthetic tissues.

4. A nucleic acid construct comprising the isolated nucleic acid of claim 2 operably linked to a recombinant nucleic acid, wherein the nucleic acid construct when introduced into leaf tissues and/or photosynthetic tissues, stimulates expression of the gene recombinant nucleic acid in the leaf tissues and/or photosynthetic tissues.

5. The isolated nucleic acid of claim 2, wherein the fragment of the nucleic acid of SEQ ID NO:1 further comprises one or more promoter elements as depicted in FIG. 12 selected from the group consisting of: initiator element (Inr), I-box, wound responsive element (WUN), abscisic acid responsive element (ABA), and heat-shock responsive element (HSE).