WO2014026312A1 - Cotton isopentenyl-transferase, gene encoding same and uses thereof - Google Patents

Description

 FIELD OF THE INVENTION The present invention relates to a prenyltransferase and a gene encoding the same, and particularly to a cotton-derived isopentenyl transferase IPT1-1 and Its coding gene, and its application in the cultivation of transgenic plants with improved drought tolerance. BACKGROUND OF THE INVENTION Abiotic stresses, such as drought, salt, extreme temperature, chemical pollution and oxygen damage, can cause serious damage to plant growth and development, and cause great losses to crop yield. The impact of drought on crop yield is It takes the first place in natural adversity, and its harm is equivalent to the sum of other disasters. It is the bottleneck of agricultural development in many areas. According to statistics, the world's dry and semi-dry areas account for 34% of the land area; China's dry and semi-arid areas account for 52% of the country's land area, and the annual area is 20 to 2.7 million hectares. About 3 billion cubic meters, due to lack of water, less than 350 to 4 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and the spring drought frequently reaches 10 years and nine times. .

Since the stress tolerance of plants is mostly quantitative, the available germplasm resources are scarce. It is very difficult to improve the stress tolerance of plants by conventional breeding techniques. It is especially difficult to cultivate true stress-tolerant varieties. In recent years, with the deepening of research on the molecular mechanism of plant stress resistance and the rapid development of molecular biology technology, stress resistance research has progressed from physiological level to molecular level, which promoted the development of plant stress resistance genetic engineering. When plants are stressed, they will respond accordingly to reduce or eliminate the damage to plants. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. These genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification and transcriptional control; (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions. In recent years, studies on the ability of plants to improve stress tolerance through transgenic techniques, as well as studies on stress-tolerant crops, xerophytes and halophytes, have yielded significant results for stress-related genes and signal transduction. The system has further advanced the corner army (Liu Q.1998. Two transcrip tion facto rs, DREBl and DREB2, w ith an EREBP/AP2 DNA binding domain, separate two cellular signal transduction Pathways in drought- and low temperature-responsive gene exp ression, respectively, in A rabidopsis. Plant Cell, 10: 1391-1406; KAN GJY.2002. A rabid op sis basic leucine zipper p ro teins that mediate stress 2responsive abscisic acid signaling. Plant Cell, 14: 343-357; ABE H.2003.A rabid op sis AtMYC2 (bHLH) and AtMYB2(MYB) function as transcrip tional activato rs in abscisic acid signaling. Plant Cell, 15: 63-78.

 However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress remain to be further studied. Studies on the function and expression regulation of stress-responsive genes are dominant, but the link between stress-resistance-related signaling pathways and the mechanism of the entire signaling network system remains to be further studied.

SUMMARY OF THE INVENTION The present inventors cloned the DNA sequence of a gene encoding a isopentenyltransferase (designated herein as IPT1-1) of cotton using SSH in combination with RACE. It was found that the introduction of the transgenic plants significantly improved the drought tolerance of the transgenic plants, and these traits were stably inherited.

 A first aspect of the invention provides a gene encoding a prenyltransferase IPT1-1 of cotton, the sequence of which is SEQ ID NO: 2.

 A second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the rd29A-GMPT1-1-2300 carrier shown in Fig. 2.

 A third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.

 A fourth aspect of the present invention provides a method for improving drought tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and causing the gene Expression; Preferably, the plant is tobacco.

A fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant comprising the gene of the first aspect of the invention, the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Tissue; Preferably, the plant is tobacco. A sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought tolerance of a plant and for use in plant breeding Use; Preferably, the plant is tobacco.

 The seventh aspect of the present invention provides the amino acid sequence encoded by the gene of the first aspect of the present invention, as shown in SEQ ID NO: 1. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (rd29A-GMPT1-1-2300) of GhIPT1-1. Figure 2 is a plasmid map of the plant expression vector (rd29A-GMPT1-1-2300) of GhIPTl-1.

 Figure 3 shows the drought tolerance simulation results of GhIPTl-1 T1 transgenic tobacco plants (in the figure, T1A1; right, T1A5) and non-transgenic tobacco plants (left, CK).

 Figure 4 shows the results of protein expression verification at the transcriptional level of transgenic T1 tobacco plants and non-transgenic control plants. M is Marker, 1_5 is the control tobacco, 6_18 is the drought-tolerant transgenic tobacco T1 plant, and 19-24 is the drought-tolerant transgenic tobacco T1 plant. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. Example 1. Cotton SSH library construction under drought stress:

 The specific method is:

 A subtractive library was constructed by inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit. The mRNA of the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a control. The specific steps are as follows:

 (1) Test materials:

冀棉14 (National Cotton Medium-term Repository, obtained by the China Cotton Research Institute, Uniform No.: ZM-30270) Seeded on sterilized vermiculite, cultured at 25 ° C, photoperiod 16 h / 8 h, poured 1 per week /2MS medium (9. 39 mM KN03, 0. 625 mM KH2P04, 10. 3 mM NH4N03, 0.75 mM MgS04, 1. 5 mM CaC12, 50 μM KI , 100 μ Μ H3B03, 100 μ Μ MnS04, 30 μM ZnS04, Ι μ Μ Na2Mo04, 0.1 μM CoC12, 100 μM Na2EDTA, 100 μ Μ FeS04). It was used for experiments when the seedlings were as high as 25-30 cm.

 (2) Material handling:

 The test seedlings were divided into two groups, each with 4 pots and 1 pot per pot. The first group was the control group, cultured at 25 ° C, lighted, and normally watered. The second group was the drought treatment group, 25 °C, light culture, stop watering, treatment for 10 days. After the treatment, the leaves of the top 1/3 of the two seedlings were cut out in time, and then rapidly frozen with liquid nitrogen at -70 °C. Store in the refrigerator.

 (3) Total RNA extraction:

 The cotton leaves of the control group and the drought treatment group were respectively taken 0.5 g, and the plant 匪 extraction kit was used.

(invitrogen) Extracts total RNA from cotton. 0%的质量。 Using HITACHI's UV spectrophotometer U-2001 to determine the total RNA absorbance at 260 nm and 280 nm, 0D260/0D280 ratio of 1. 8-2· 0, indicating that the total RNA purity is higher, with 1.0% The integrity of total RNA was detected by agarose gel electrophoresis, and the brightness of the 28S band was approximately twice that of the 18S band, indicating good RNA integrity. The mRNA was isolated using Qiagen's Oligotex mRNA purification assay [JP (purification of polyA+ RNA from total RNA).

 (4) Inhibition subtractive hybridization:

 In order to gain the effectiveness of the Expressed Sequence Tag (EST) (unigene), the gene is not cleavable and the obtained sequence is in the untranslated region. The laboratory uses Rsal, Haelll to digest the double-stranded cDNA (according to the protocol described in the subtractive kit, obtained by reverse transcription of mRNA), and performs two sets of suppression subtraction. Other steps and methods are based on Clontech's PCR-selectTM. The method shown in the cDNA Subtraction Kit was used for suppression subtractive hybridization, and finally the second PCR product of the two groups of forward subtracted hybrid cDNA fragments was combined.

 (5) Construction and preliminary screening, cloning and identification of cDNA subtractive library

 Forward subtraction of the second PCR product of the hybrid cDNA fragment (QIAquick PCR Purification)

Kit purified, purchased from Qiagen) and pGEM-T Easy (purchased from Promega kit) vector, according to the procedure of pGEM-T Easy kit, the specific steps are as follows: Add the following components in sequence with 200 μ 1 PCR tube: Purified positive The second PCR product of the subtractive hybridization cDNA fragment was 3 μl, Τ4 ligase buffer 5 μl , pGEM-T Easy vector 1 μl, Τ4 DNA ligase 1 μΐ, and ligated overnight at 4 °C. Take 10 linked reaction products and add to 100 competent E. coli JMI09 (purchased from In TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, plus 250 L LB medium

(1% Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm). In a 37 °C water bath, cultured at 225 r/min for 30 min, 200 μL of bacterial solution was planted. Yu Han

LB (ibid.) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) of 50 μg/mL ampicillin was cultured on a culture plate at 37 °C. 18 counts of clear white and blue with a diameter > 1 mm in the culture plate. The number of colonies was collected, and 360 white colonies were randomly picked (number: Gh-D001 to Gh-D360). All white clones were picked in 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 μg/mL ampicillin, and cultured overnight at 37 °C, glycerol was added to a final concentration of 20%, and stored at -80 °C. spare. The nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-se ectTM cDNA Subtraction Ki t kit) were used for PCR amplification, and 292 positive clones were obtained, and all positive clones were sent to the UK.潍Jieji (Shanghai) Trading Co., Ltd. sequencing

 (6) cDNA sequencing analysis of differential clones:

 After DNA sequencing results were removed from the vector and the ambiguous sequence and redundant cDNA, a total of 180 ESTs (uni genes) were obtained. BlastN found that 102 uni genes have homologous sequences in GenBank (protein homology is 50% or more). 33 EST functions are unknown or hypothetical proteins, and another 45 have not obtained homologous matches, presumably a shorter sequence at the 3 ', 5 ' untranslated region. Example 2 Cloning of cotton isopentenyl transferase encoding gene GhIPT l-1

 After the cloned Gh-D21 1 was detached from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence belonged to the isopentenyl transferase, and the full length of the clone Gh-D21 1 was encoded herein. The gene was named GhIPT l-l, and the corresponding protein was named IPT 1- 1.

 SEQ

 1 ACAACCCCGA GTGAATTTGG GGCTCAACCT GGAGACTATC TTCCGCAGAA AGGATAAGGT

61 GGTGTTTGTA ATGGGAGCAA CCGGCACCGG CAAGTCCCGT CTGGCAATCG ATCTGGCCAC

121 CCATTTTCCA GCTGAAATTG TAAATTCAGA CAAAATGCAA GTATATAAAG GCCTTGACAT

181 ATTAACTAAC AAAGTCACTG TAGAGGAGTG CCGTGGGGTT CCACACCATT TGCTAGGCAT

241 AGTAGACCCA ATCTCCAATT TCACATCCAT GGATTTCCAG CATCATGCTT CACTTGCTGT

301 TGAATCCATA CTTGCGGAAG GCCGGCTCCC AATCATCGCC GGTGGCTCCA ATTCATACAT

361 CGAGGCATTG GTGAATGATG ACCCTGAATT TCAATTAAGA TACGAATGCT GCTTCCTATG

421 GGTTGATGTG TCATTGCGCG TGTTACACTC CTTTGTGTCG GAACGGGTAG ATAAGATGGT

481 GAAAGCAGGC TTGGTAGATG AGGTGGAACA AATGTTTGAT CCAATGGCTG ATTATTCCCG

541 AGGGATTAGG CGTGCGATTG GGGTCCCCGA ATTGGACCAA TATTTCCGTG CTGGATCAAT

601 TCTTGATGCC AACATTCGAG CTAGGATGCT TGACACGGCC ATTTCAAAAA TCAAGGAAAA

661 TACATGCACT CTAGCTTCTC GCCAGTTCCA TAAAATCTGC CGCCTTTATA ACCAACGGAA 721 TTGGAGAATG CACCGGATTG ATGCCACCGA AGTTTTCCTA AAGCGTGGCG ATGAGGCCGA

781 TGAGACATGG GAGAGACTTG TTGCGGGACC AAGCACCATG ATAGTGGACA AATTCCTCTT

841 TGAAAAAAAT CGTGTGCCCA CCATTTTGCC GTCTACCGCC TCATCATCGG TCCCTATCGC

901 CGCCGTAGCA GCCGCATCCC GTTAAAGGTA GGACCCGTAG AATCTCACAC ATGCAAATGA

961 TGTCCCACGT GGCATCACGC CACATGGCAG GCTGGCATCC TATACCTCTC TCTCTCTACA

1021 TATTTTTTAT CTGAATTTGT TTTTCAATGA TTAGTCAATA GTCATAATGA AAAAAATAAA

1081 GAAAAATAGT TTATGGGTTG GAGATCGGAG AAGTTATAGT TGTAGACGTT GCTGCGAGCC

1141 CGGTCGTTAA ACTGTGT

Cloning of the full-length gene of GhIPTl-1

 The GhIPTl-1 gene fragment that has been obtained already has a stop codon TAA, and only 5 ' RACE is required. Based on the obtained Gh-D211 gene fragment, three specific primers were designed as reverse transcription primers and 3'-end specific primers for 5' RACE.

 GhIPTl-1 GSP1 : SEQ ID NO : 4:

 CCCATGTCTC ATCGGCCTCA

 GhIPTl-1 GSP2: SEQ ID NO: 5:

 GGCGAGAAGC TAGAGTGCAT GTA

 GhIPTl-1 GSP3: SEQ ID NO: 6:

 GGCATCAAGA ATTGATCCAG CACG

 The experimental procedure was performed according to the kit instructions (5, RACE System for Rapid Amplification of cDNA Ends kit was purchased from Invitrogen).

The first round of PCR amplification was carried out using SEQ ID NO: 5 and 5 ' universal primer AAP (provided with the kit), and mRNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 4) as a template, the specific steps are as follows: Ex Taq was purchased from TAKARA, 50 μ 1 PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 mRNA reverse transcribed cDNA, 1. 0 μ 1 Ex Taq 10 μM of primers SEQ ID NO: 5 and AAP each of 2.0 μl, and 35 μl of double distilled water. PCR conditions: 94 ° C denaturation for 5 min, 94 ° C denaturation 30 s, 55 ° C annealing 30 s, 72 ° C extend 2min, after 33 cycles, 72 ° C extend 10 min ₀ The resulting PCR product was double After diluting 50 times of distilled water, take 2.0 μl as template and carry out the second round of PCR amplification with SEQ ID NO: 6 and 3' primer AUAP. The specific steps are as follows: 50 μ 1 PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 diluted first round PCR product, 1. 0 l Ex Taci, 10 μM primer SEQ ID NO: 6 and AUAP each 2. 0 μ 1, and 35 μ 1 of double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, Anneal at 58 °C for 30 s, 72 °C for 2 min, after 33 cycles, and at 72 °C for 10 min. The second PCR product was recovered from a fragment of about 700 bp (Gel Extraction Kit was purchased from OMEGA). It was ligated to pGEM-T Easy Vector, transformed into JM109 (specifically the same as above), and 10 white colonies were randomly picked up to contain 50 g/mL. Ampicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20%, and stored at -80 ° C until use. SEQ ID NO: 6 and 3' primer AUAP were used for PCR amplification (reaction system and reaction conditions are the same as above), and three positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing. The 5' end of the cDNA. The obtained 5' RACE product was cloned and sequenced, and spliced with the cloned Gh-D211 sequencing result.

SEQ ID NO: 17:

 1 GAAGCAGATT TTGCAACTGG TTGTGTTCTT CCACTTTTGC AGGGTTTAAG ATGAAACTTT

61 CTATGTCTGC CAGCAAACTA GTACAACCCC GAGTGAATTT GGGGCTCAAC CTGGAGACTA

121 TCTTCCGCAG AAAGGATAAG GTGGTGTTTG TAATGGGAGC AACCGGCACC GGCAAGTCCC

181 GTCTGGCAAT CGATCTGGCC ACCCATTTTC CAGCTGAAAT TGTAAATTCA GACAAAATGC

241 AAGTATATAA AGGCCTTGAC ATATTAACTA ACAAAGTCAC TGTAGAGGAG TGCCGTGGGG

301 TTCCACACCA TTTGCTAGGC AT AG T AG AC C CAATCTCCAA TTTCACATCC ATGGATTTCC

361 AGCATCATGC TTCACTTGCT GTTGAATCCA TACTTGCGGA AGGCCGGCTC CCAATCATCG

421 CCGGTGGCTC CAATTCATAC ATCGAGGCAT TGGTGAATGA TGACCCTGAA TTTCAATTAA

481 GATACGAATG CTGCTTCCTA TGGGTTGATG TGTCATTGCG CGTGTTACAC TCCTTTGTGT

541 CGGAACGGGT AGATAAGATG GTGAAAGCAG GCTTGGTAGA TGAGGTGGAA CAAATGTTTG

601 ATCCAATGGC TGATTATTCC CGAGGGATTA GGCGTGCGAT TGGGGTCCCC GAATTGGACC

661 AATATTTCCG TGCTGGATCA ATTCTTGATG CCAACATTCG AGCTAGGATG CTTGACACGG

721 CCATTTCAAA AATCAAGGAA AATACATGCA CTCTAGCTTC TCGCCAGTTC CATAAAATCT

781 GCCGCCTTTA TAACCAACGG AATTGGAGAA TGCACCGGAT TGATGCCACC GAAGTTTTCC

841 TAAAGCGTGG CGATGAGGCC GAT GAG AC AT GGGAGAGACT TGTTGCGGGA CCAAGCACCA

901 TGATAGTGGA CAAATTCCTC TTTGAAAAAA ATCGTGTGCC CACCATTTTG CCGTCTACCG

961 CCTCATCATC GGTCCCTATC GCCGCCGTAG CAGCCGCATC CCGTTAAAGG TAGGACCCGT

1021 AGAATCTCAC ACATGCAAAT GATGTCCCAC GTGGCATCAC GCCACATGGC AGGCTGGCAT

1081 CCTATACCTC TCTCTCTCTA CATATTTTTT ATCTGAATTT GTTTTTCAAT GATTAGTCAA

1141 TAGTCATAAT GAAAAAAATA AAGAAAAATA GTTTATGGGT TGGAGATCGG AGAAGTTATA

1201 GTTGTAGACG TTGCTGCGAG CCCGGTCGTT AAACTGTGT A pair of primers were designed according to the sequence of SEQ ID NO: 17 as follows:

 GhIPTl-lF: SEQ ID NO: 7:

 ATGAAACTTTCTATGTCTGCCAGCA

 GhIPTl-lR: SEQ ID NO: 8:

TTAACGGGATGCGGCTGCTACGG The full length of GhIPTl-1 was cloned by SEQ ID NO: 7 and SEQ ID NO: 8.

 The PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template. 50 μ 1 PCR reaction system: 10 μΐ 5XPS Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR, 10 μM primer SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 μ 1, and 30 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.

 The PCR amplification product was added with A tail: The PCR product was added 2.5 times of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 μl of double distilled water. Add 2.5 μl 10 X Ex Buffer, 0.5 μl 5 mM dATP, 2.5 μ 1 ΙΟΧΕχ Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 900 bp was recovered (Omega Recovery Kit), ligated into the pGEM T-easy vector, transformed into JM109 (method as above), and 10 white colonies were randomly picked from LB containing 50 μg/mL ampicillin. Incubate in liquid medium, incubate at 37 ° C overnight, add glycerol to a final concentration of 20%, and store at -80 °C until use. SEQ ID NO: 7 and SEQ ID NO: 8 were used for bacterial liquid PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The sequence is SEQ ID. NO: 2.

 Amino acid sequence of IPT1-1 protein: SEQ ID NO: 1

 1 MKLSMSASKL VQPRVNLGLN

 21 LETIFRRKDK WFVMGATGT

 41 GKSRLAIDLA THFPAEIVNS

 61 DKMQVYKGLD LTNKVTVEE

 81 CRGVPHHLLG IVDPISNFTS

 101 MDFQHHASLA VESILAEGRL

 121 PIIAGGSNSY EALVNDDPE

 141 FQLRYECCFL WVDVSLRVLH

 161 SFVSERVDKM VKAGLVDEVE

 181 QMFDPMADYS RGIRRAIGVP

 201 ELDQYFRAGS Worker: LDANIRARM

 221 LDTAISKIKE NTCTLASRQF

 241 HKICRLYNQR hidden RMHRIDAT

 261 EVFLKRGDEA DETWERLVAG

 281 PSTMIVDKFL FEKNRVPT: L

 301 PSTASSSVPI AAVAAASR*

Nucleotide sequence of the GhIPTl-1 encoding gene: SEQ ID NO: 2

 1 ATGAAACTTT CTATGTCTGC CAGCAAACTA GTACAACCCC GAGTGAATTT GGGGCTCAAC

61 CTGGAGACTA TCTTCCGCAG AAAGGATAAG GTGGTGTTTG TAATGGGAGC AACCGGCACC 121 GGCAAGTCCC GTCTGGCAAT CGATCTGGCC ACCCATTTTC CAGCTGAAAT TGTAAATTCA

181 GACAAAATGC AAGTATATAA AGGCCTTGAC ATATTAACTA ACAAAGTCAC TGTAGAGGAG

241 TGCCGTGGGG TTCCACACCA TTTGCTAGGC AT AG T AG AC C CAATCTCCAA TTTCACATCC

301 ATGGATTTCC AGCATCATGC TTCACTTGCT GTTGAATCCA TACTTGCGGA AGGCCGGCTC

361 CCAATCATCG CCGGTGGCTC CAATTCATAC ATCGAGGCAT TGGTGAATGA TGACCCTGAA

421 TTTCAATTAA GATACGAATG CTGCTTCCTA TGGGTTGATG TGTCATTGCG CGTGTTACAC

481 TCCTTTGTGT CGGAACGGGT AGATAAGATG GTGAAAGCAG GCTTGGTAGA TGAGGTGGAA

541 CAAATGTTTG ATCCAATGGC TGATTATTCC CGAGGGATTA GGCGTGCGAT TGGGGTCCCC

601 GAATTGGACC AATATTTCCG TGCTGGATCA ATTCTTGATG CCAACATTCG AGCTAGGATG

661 CTTGACACGG CCATTTCAAA AATCAAGGAA AATACATGCA CTCTAGCTTC TCGCCAGTTC

721 CATAAAATCT GCCGCCTTTA TAACCAACGG AATTGGAGAA TGCACCGGAT TGATGCCACC

781 GAAGTTTTCC TAAAGCGTGG CGATGAGGCC GAT GAG AC AT GGGAGAGACT TGTTGCGGGA

841 CCAAGCACCA TGATAGTGGA CAAATTCCTC TTTGAAAAAA ATCGTGTGCC CACCATTTTG

901 CCGTCTACCG CCTCATCATC GGTCCCTATC GCCGCCGTAG CAGCCGCATC CCGTTAA Example 3 Construction of GhIPTl-1 gene plant expression vector

 The plant binary expression vector PCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as the plant expression vector, and the 35S promoter of the ΝΡΤΠ gene containing the double enhancer was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The inducible promoters rd29A and Tnos were selected as promoters and terminators of the GhIPTl-1 gene.

 Pnos were amplified using the plant expression vector PBI 121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using primers SEQ ID NO: 9 and SEQ ID NO: 10, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 1. 0 μ 1 PBI 121 , 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO : 9 And SEQ ID NO: 10 each of 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min. PCAMBIA2300-1 was obtained by EcoRI, Bglll digestion with pCAMBIA2300 (promega, T4 ligase cassette).

 SEQ ID NO: 9:

 GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO : 10:

 ATCCAGATCTAGATCCGGTGCAGATTATTTG

SEQ ID NO: 11 and SEQ ID NO: 12 amplify Tnos using PBI 121 as a template, using PrimeSTAR HS DNA polymerase of TaKaRa. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μl 2.5 mM dNTP, 1.0 μl PBI121, 1.0 μl PrimeSTAR, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 μl, and 31 μl of double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min. pCAMBIA2300-2 was obtained by restriction enzyme digestion with pCAMBIA2300-l (promega T4 ligase cassette) by Sacl and EcoRI

 SEQ ID NO: 11:

 AAG dOTAATTTCCCCGATCGTTCAAA

 SEQ ID NO: 12:

 TCA GAA mCCAGTGAATTCCCGATCTAGTA

 SEQ ID NO: 13 and SEQ ID NO: 14 The Arabidopsis rd29A promoter was amplified using Arabidopsis thaliana (Columbia type, available from www. arabidopsis.org) DNA as a template (see Zeng J., et L. 2002, Preparation). Of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44(6): Method 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 μ 1 PCR reaction system: 10 μ 15×PS Buffer, 3 μ 1 2· 5 mM dNTP, 1.0 μ 1 Arabidopsis DNA, 1.0 μ 1 PrimeSTAR, 10 μ Μ primers SEQ ID NO: 13 and SEQ ID NO: 14 each of 2.0 μ 1, and 31 μ 1 of double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min by HindIII, Pstl digestion Connected to (connection method is the same as above) pCAMBIA2300-2 obtained pCAMBIA2300_3

 SEQ ID NO: 13:

 ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO: 14:

 TGAO^ CTCCAAAGATTTTTTTCTTTCCAATAG

SEQ ID NO: 15 and SEQ ID NO: 16 amplify GhIPTl-1 (template is GhIPTl-1 obtained in Example 2), using PrimeSTAR HS DNA polymerase of TaKaRa. 50 μ 1 PCR reaction system: 10 μ 15×PS Buffer, 3 μ 12· 5 mM dNTP, 1.0 μ 1 GhlPTl- 1- pGEM, 1.0 μ 1 PrimeSTAR, 10 μM primers SEQ ID NO: 15 and SEQ ID NO: 16 each of 2·0 μ 1, and 31 μ 1 of double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. By Pstl, Sacl The enzyme expression vector rd29A_GhlPTl-1-2300 was obtained by restriction enzyme ligation (connection method as above) PCAMBIA2300-3.

 SEQ ID NO: 15:

 TGAO^ TGAAACTTTCTATGTCTGCCAGCA

 SEQ ID NO : 16:

 AAG dOmAACGGGATGCGGCTGCTACGG Example 4 rd29A-GhIPTl-1230 expression vector for transformation of Agrobacterium

Agrobacterium LBA4404 (available from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was plated on LB solid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin 1-2 days in advance Single spot inoculation, culture at 28 ° C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin. Incubate overnight at 28 °C (about 12-16 h) to 0D600 value. 0. 4, forming a seed bacterial liquid. 5小时至0D _6。 5 ml of activated bacterial solution (1: 20 ratio) was inoculated in 100 ml of the same concentration of antibiotics in LB liquid medium, shaking at 2 ° C. 2 h. . =0. 8. The ice bath solution was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 min at 4 °C, discard the supernatant; add a certain amount of pre-cooled 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 °C, collect the precipitate; repeat wash 3-10 times with 10% glycerol Add the appropriate amount of ice bath pre-cooled 10% glycerol to resuspend the bacterial pellet, dispense it in 40 μl/tube, and store at -70 °C until use.

Transformation of Agrobacterium: Thaw competent cells on ice, add 1 μl of plasmid to 40 μl of competent cells, mix and mix with ice bath for about 10 mino. Transfer the mixture of competent and DNA to pre-cooled by gun. In the electric shock cup, tap to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from bio-rad) on the slide of the electric shock chamber and push the slide to place the electric shock cup on the base electrode of the electric shock chamber. When using a 0. 1cm electric shock cup, the program of MicroPulser (purchased from bio_rad) is set to "Agr" and the electric shock is once. The electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently spread the cells with a gun. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h. Take 100~200 μ ΐ of the bacterial solution and plate on the corresponding resistant screening medium (LB solid medium containing 50 μg/ml rifampicin, 50 μg/ml streptomycin, 50 μg/ Ml Kanamycin), cultured at 28 °C. Example 5 Obtaining Transgenic Tobacco by Agrobacterium-mediated Transformation

To soak tobacco seeds with 75% alcohol (National Tobacco Medium Term Bank, obtained by the Institute of Tobacco, Chinese Academy of Agricultural Sciences, bank number I5A00660) 30 s, washed twice with sterile double distilled water. Then, it was immersed in 0.1% liter of mercury for 8 minutes, and washed twice with sterilized double-distilled water to complete surface sterilization. Surface-sterilized tobacco seeds were placed in MS ( 18.78 mM KN0 ₃ , 1. 25 mM KH ₂ P0 ₄ , 20. 6 mM NH^Oa, 1. 5 mM MgS0 ₄ , 3. 0 mM CaCl ₂ , 50 μ M KI , 100 μ M H3BO3, 100 M MnS0 ₄ , 30 μ M ZnS0 ₄ , 1 μ M Na ₂ Mo0 ₄ , 0.1 M CoCl ₂ , 100 μ M N3⁄4EDTA, 100 μ M FeS0 ₄ , 7. 4 g /L agar, sucrose 30 g / L) was germinated under aseptic conditions to prepare sterile seedlings. The leaves of the sterile seedlings were cut into 5 mm×5 hidden leaf discs, and the leaf discs were inoculated with the Agrobacterium containing the expression vector rd29A-GhIPTl-l-2300 in the logarithmic growth phase for 10 min, and the bacterial liquid was sucked in the dark condition. The cells were cultured for 2 days (MS medium). Transfer leaves to differentiation medium (MS+1 mg/L cytokinin (BA) +0.1 mg/L naphthaleneacetic acid (NAA) +50 mg/L kanamycin + 500 mg/L cephalosporin) On the upper and lower culture conditions for about 45 days, after the buds grow up, they are transferred to rooting medium (MS + 50 mg / L kanamycin + 500 mg / L cephalosporin) for about 30 days, until the root system After development, the seedlings were transferred to MS medium supplemented with 500 mg/L cephalosporin for number storage.

The obtained transgenic tobacco leaves were taken, DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and SEQ ID NO: 7 and SEQ ID NO: 8 (50 μl PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer) , 3 μ 1 2 · 5 mM dNTP, 2. 0 μ 1 DNA, 1. 0 μ 1 Ex Taq 10 μ M primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2. 0 μ 1, and 35 Double distilled water of μ 1. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C 10 Min), PCR identification, preservation of positive plants for number T. A1_T. A20. Example 6 Drought Tolerance Simulation Experiment and Functional Identification of GhIPTl-1 Transgenic Tobacco T1 The sterilized vermiculite was soaked in 1/2 MS medium. T. A1-T. A20 and control tobacco seeds were sown on vermiculite, 15 seeds per pot, 25 ° C, 10 hours light culture / 14 hours dark culture cycle, 1/2 MS every 5 days, 25 days after culture, each pot was kept 4_5 seedlings of uniform size, drought test, transgenic tobacco, control tobacco drought for 14 days (no watering), 25 ° C, 10 hours light culture / 14 hours dark culture cycle. The drought resistance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the control plants were wilting, while Ί Α 1, Ί Α 、 、 、 Α Α Α Α Α 、 、 Α Α Α Α Α Α Α Α Α Ί Ί Ί Α17, Ί\Α19 Seven out of a total of 32 tobacco plants were able to grow normally, showing obvious drought tolerance (see Figure 3, taking Ί\Α1, Ί\Α5 as an example, Ί\Α8, Ί\ The results of Α13, Ί\Α15, Ί\Α17, Ί\Α19 are similar to Ί\Α1, Ί\Α5, not shown here). Example 7 Verification of IPTl-1 protein expression at the transcriptional level

 Control tobacco, drought-tolerant transgenic tobacco T1 plants, drought-tolerant transgenic tobacco T1 plants (good growth) drought 14 days leaves 0. 05g, total RNA extracted by plant RNA extraction kit (invitrogen). The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations. Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription kit Superscript I I I Reverse Transcriptase (2 μg total RNA as a template, reverse transcription primer SEQ ID NO: 8). The relative expression of IPT1-1 protein was detected by augmenting GhIPT1-l with SEQ ID NO: 7 and SEQ ID NO: 8. PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO: 7 and SEQ ID NO: 8 each of 2.0 μl, and 30 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, after 29 cycles, extension at 72 °C for 10 min. The electrophoresis results of the product are shown in Figure 4: M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-5 is control tobacco, and 6-18 is drought-tolerant transgenic tobacco T1 plant, 19-24 It is a T1 generation plant that is not tolerant to transgenic tobacco. The strip size shown in the figure is the same as the size of GhIPTl-1. The results showed that the control tobacco did not transcribe the mRNA of GhIPTl-1, and the T1 generation of drought-tolerant transgenic tobacco had stronger transcription to GhIPTl-1, and the T1 generation of the drought-tolerant transgenic tobacco had no transcription or transcription.

Claims

Claim A cotton isoprenyltransferase encoding gene having the sequence of SEQ ID NO: 2.  2. A recombinant expression vector comprising the gene of claim 1 and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector.  3. The vector of claim 2 which is the rd29A-GMPT1-1-2300 carrier shown in Figure 2. A recombinant cell comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3; preferably, the recombinant cell is a recombinant Agrobacterium cell.  A method for improving drought tolerance of a plant, comprising: introducing the gene of claim 1 or the recombinant expression vector of claim 2 or 3 into a plant or plant tissue and expressing the gene; preferably, The plant is tobacco.  A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3 under conditions effective to produce a plant.  7. The method of claim 6 wherein the plant is tobacco.  The gene of claim 1, the recombinant expression vector of claim 2 or 3, or the recombinant cell of claim 4 for use in improving drought tolerance of a plant and for use in plant breeding.  9. The use of claim 8 wherein the plant is tobacco.  The gene-encoded protein according to claim 1, which has the sequence shown in SEQ ID NO: 1.