WO2014172847A1 - Myb transcription factor myb1-2 of thellungiella salsuginea and coding gene, and use thereof - Google Patents

Abstract

Disclosed are a MYB transcription factor MYB1-2 derived from Thellungiella salsuginea and a coding gene thereof, and the use thereof in the cultivation of transgenic plants having improved drought tolerance.

Description

 TECHNICAL FIELD The present invention relates to a MYB transcription factor and a coding gene thereof, and particularly to a MYB transcription factor MYB 1-2 derived from salt mustard. And its coding genes, as well as their use 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, causing great losses to crop yield, and 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-arid areas account for 34% of the land area; China's dry and semi-arid areas account for about 52% of the country's land area, and the annual drought area amounts to 200-2.7 million hectares. 30 billion cubic meters, due to lack of water, the amount of food is reduced by 35 to 40 billion kilograms; especially China's main grain-producing areas such as North China, Northeast China and Northwest China are the areas with the most water shortage in China, and the spring drought frequently reaches 10 years.

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, and it is particularly 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 systems 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 a further understanding (Liu Q.1998. Two transcription factors, DREB 1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought And low temperature responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10: 1391-1406; KANGJY.2002. Arabidopsis basic leucine zipper proteins that mediate stress responsive abscisic acid signaling. Plant Cell, 14: 343-357; ABEH.2003.A rabid op sis AtMYC2 (bHLH) and AtMYB2(MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78.

 In the plant stress response system, the regulation of functional gene expression by transcription factors is a key link in plant stress response. As one of the largest families of transcription factors in plants, MYB transcription factors play an important role in plant stress tolerance. 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 will provide an important basis for the link between plant stress-resistance-related signaling pathways and the entire signaling network system. SUMMARY OF THE INVENTION The present inventors cloned a MYB transcription factor of salt mustard using SSH (Suppression Subtractive Hybridization) in combination with RACE (rapid amplification of cDNA ends) (the DNA sequence of the coding gene designated herein) and found that When introduced into recipient plants, the drought tolerance of transgenic plants can be significantly improved, and these traits can be stably inherited.

 The first aspect of the present invention provides a gene encoding a MYB-like transcription factor MYB1-2 of salt mustard (herein named Γ/ζ Β -2) having the sequence of SEQ ID NO: 2.

 A second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, preferably, the expression vector is pCAMBIA2300; And the nucleotide sequence of the gene is operably linked to the expression control sequence of the recombinant expression vector; preferably, the recombinant expression vector is the rd29A-ThMYB 1-2-2300 vector shown in Figure 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 a 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.

A seventh aspect of the invention provides the amino acid sequence encoded by the gene of the first aspect of the invention, as set forth in SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a construction flow of a plant expression vector (rd29A-ThMYB 1-2-2300) of ThMYB1-2. Figure 2 is a plasmid map of the plant expression vector (rd29A-ThMYB 1-2-2300) of ThMYBl-2. Figure 3 shows the results of drought tolerance simulation experiments of Γ3⁄4 -2 Το transgenic tobacco plants (T _Q C _{3 ;} right, T _Q C ₉ ) and non-transgenic tobacco plants (left CK) as controls.

Figure 4 is a verification result of molecular level detection of the Γ Λ 3⁄4 -2 gene in T1 transgenic tobacco plants and non-transgenic control plants by reverse transcription PCR at the transcriptional level. M is DNA Ladder Marker (DL2000, TakaRa), 1-4 are non-transgenic control tobacco plants, 5-20 are transgenic tobacco T _Q plants with significant drought tolerance, and 21-24 are transgenic tobacco T with insignificant drought tolerance. _Q generation plants. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples.

 The unrecognized restriction enzymes mentioned in the examples below were purchased from New England Biolabs. Example 1. Salt mustard SSH library construction under drought stress:

 The specific method is:

A method according to PCR-select ^TM cDNA Clontech kit Subtraction Kit's instructions shown by suppression subtractive hybridization subtraction library was constructed (SSH library). Growing during the experiment The mRNA of the leaves of the salt-treated mustard seedlings in the process of drought treatment was used as a sample (Tester), and the mRNA of the leaves of the untreated salt mustard seedlings was used as a control. Specific steps are as follows:

 (1) Test materials:

Salt mustard seeds (purchased at the Yanlan Plant Breeding Center of Wulanbu and Desert Green Botanical Garden in Bayannao, Inner Mongolia, China) are planted on sterilized vermiculite at 16 °C, photoperiod 16 hours light/8 hours dark (light Incubate under conditions of 2000-3000 Lx), and pour 1/2MS medium per week (9.39 mM KN0 ₃ , 0.625 mM KH ₂ P0 ₄ , 10.3 mM H ₄ N0 ₃ , 0.75 mM MgS0 ₄ , 1.5 mM CaCl ₂ , 50 μΜ ΚΙ, 100μΜ Η ₃ ΒΟ ₃ , 100 M MnSO ₄ , 30 M ZnSO ₄ , 1μΜ Να ₂ Μο0 ₄ , Ο. ΙμΜ CoCl ₂ , ΙΟΟμΜ Na ₂ EDTA, 10(^M FeSO ₄ ) — times. When the seedling diameter is reached Used for experiments at 5-6 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 a control group, cultured at 25 °C, 16 hours light/8 hours dark, and normally 1/2MS watered. The second group was the drought treatment group, cultured at 25 °C, cycle 16 hours light/8 hours dark condition, stopped watering, treated for 10 days, then cut the leaves of the two groups in time, and quickly frozen with liquid nitrogen, at -70 °C is stored in the refrigerator.

 (3) Total RNA extraction:

The total mustard leaves of the salt mustard were extracted with the plant RNA extraction kit (Invitrogen) by taking 0.5 g of the salt mustard leaves of the control and drought treatment groups, respectively. The absorbance of total RNA at 260 nm and 280 nm was determined by HITACHI's UV spectrophotometer U-2001. The OD ₂₆₀ / OD ₂₈₀ ratio was 1.8-2.0, indicating a high total RNA purity with a 1.0% agarose gel. The total RNA was detected by electrophoresis, and the 28S band was about twice as bright as the 18S band, indicating good RNA integrity. mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purified polyA+ RNA from total RNA).

 (4) Suppression of subtractive hybridization:

The method according to Clontech's PCR-select ^TM cDNA Subtraction Kit kit instructions will be shown suppression subtractive hybridization. Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and subtractive hybridization was performed using 2 Tester cDNA and 2 μg of Driver cDNA as starting materials. The Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 hours in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated. Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization. Mixing the products of the two first subtractive hybridizations with the new denatured The Driver cDNA was subjected to a second positive subtractive hybridization, and then the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.

 In order to increase the availability of Expressed sequence tag (EST) (unigene), avoid gene-free cleavage sites and the obtained sequences in the untranslated region, Rsal, Haelll used the above procedure for Tester cDNA and Driver. The cDNA was digested and subjected to two forward subtractive hybridizations and two inhibitory PCR amplifications, and finally the second PCR products of the two sets of forward subtractive hybridization cDNA fragments were combined.

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

 The second PCR amplification product of the combined forward subtractive hybridization cDNA fragment was purified according to the method described in the product specification of pGEM-T Easy kit (purchased from Promega kit) (purified by QIAquick PCR Purification Kit, purchased from QIAquick PCR Purification Kit) Qiagen) is ligated to the pGEM-T Easy vector. The specific steps are as follows: The following components are sequentially added using a 200 μΐ PCR tube: The second PCR product of the purified positive subtractive hybridization cDNA fragment 3 μl, 2χΤ4 ligase buffer 5 l , pGEM-T Easy vector 1 μ1, Τ4 ϋΝΑ ligase Ι μΐ , ligated overnight at 4 °C. Then, 10 reaction products were taken and added to 100 competent E. coli JMI09 (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, ice bath for 2 minutes, and 250 L LB medium (1% Tryptone was purchased from OXOID, 0.5% yeast extract (Yeast Extract, purchased from OXOID), 1% NaCl (purchased from Sinopharm)), placed in a 37 ° C shaker, shaken at 225 rpm for 30 minutes, and then taken 200 μL from The bacterial solution was coated with 50 g/mL ampicillin, 40 g/mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), 24 g/mL IPTG (isopropyl LB (β-D-thiogalactopyranoside) (X-gal/IPTG purchased from TAKARA) was incubated on a solid culture plate at 37 ° C for 18 hours. Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly select 330 white colonies (number: Th-D001 to Th-D330). The picked white clones were inoculated separately into 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 g/mL ampicillin, and cultured overnight at 37 ° C, and glycerol was added to a final concentration of 20% by volume. Store at -80 ° C for later use. The cultured colonies were cloned with nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit), and 272 positive clones were obtained, and then all positive clones were obtained. Sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for 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 167 were obtained. Expressed sequence tag (EST) (Unigene). It was found by BlastN that 112 ESTs have homologous sequences in GenBank (protein homology is more than 50%), 29 EST functions are unknown or hypothetical proteins, and 35 ESTs have not obtained homologous matches, presumably at 3 ', 5, a shorter sequence of untranslated regions at the ends. Example 2 Cloning of the MYB transcription factor encoding gene ThMYBl-2 from salt mustard

 After removing the redundant DNA from the cloned colony Th-D118 clone in the identified salt mustard SSH library, the sequence is SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence is higher than the MYB transcription factor. For the homology, the full-length gene encoded by the clone Th-D118 was named as ThMYBl-2, and the corresponding protein was named ΜΥΒ1-2.

 SEQ ID No: 3

 1 GACTGAGATG GATTAATTAC CTTCGTCCTG ATCTCAAACG TGGAAACTTC TCTGATGAAG

61 AAGACCAAGT CATCATCAAA CTCCATAGCT TGCTCGGCAA CAAATGGTCA TTGATCGCTG 121 GAAGATTACC AGGAAGAACA GATAATGAAA TCAAAAACTA TTGGAACACT CATATTAAAA 181 GAAAGCTTCT TAGCCATGGA ATCGATCCAC AAACTCATCG TCCGGTTAAA GATTCCGTCG 241 CCGTTACTGA TTCCAAAACG GTTCCGTCTC CGTTAACACA CGCTCACCAA AACGGCGCCG 3 01 TTGAATACTC TTTCTCCGTT AGACCTAAGA AGGAAATTTC CTCCGATAAC GGCGCTTCCA 361 CAAGCGGCAC GACGACCGAT GAGGATCTCC AGCAGAACGG CGATTGTTAC TACAGTGATG 421 ATTCACGAGA CACAGAGCTG AATCTGGAGT TAACTCTCGG GTCCGGGTCA ACTTCGTGGG 481 TTGGGTCGGA TCGGGTAGTG AGAGCTGGGT TATCGGCAGA TTCGAAGCCG TGGCGGGATC 541 CGGTGACCGC GGTGGTGGCG GCGCGTTTGT CGTTGTTGTA AGAATTGCTA AACGGCAAAA 601 ATCCCCTTTA ACAAAATATC AAAA

 Cloning of the full-length gene of ThMYBl-2

 The ThMYBl-2 gene fragment (SEQ ID NO: 3) that has been obtained already has a stop codon TAA, and only 5 ' RACE is required.

 Based on the obtained ThMYBl-2 gene fragment (SEQ ID NO: 3), three specific primers were designed as reverse transcription primers and 3'-end specific primers for 5' RACE.

 ThMYBl-2 GSP1 : SEQ ID NO: 4:

 CTTGTGGAAGCGCCGTTATC

 ThMYBl-2 GSP2: SEQ ID NO: 5:

 CAACGGCGCCGTTTTGGTG

 ThMYB 1 -2 GSP3: SEQ ID NO: 6:

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

 Using the SEQ ID NO: 5 and 5' universal primer AAP (provided with the kit), the reverse transcription cDNA (reverse transcription primer SEQ ID NO: 4) extracted from the leaves of the drought-treated group was used as a template for the first round of PCR amplification. Increase, the specific steps are as follows:

 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq (Ex Taq from TAKARA), 10 μΜ primer SEQ ID NO: 5 and AAP is 2.0 μl each, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.

 The obtained PCR product was diluted 50 times with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 6 and the 3' primer AUAP. The specific steps are as follows:

 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 l Ex Taq, 10 μΜ primer SEQ ID NO: 6 and AUAP 2.0 μl each, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, after 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes. A band of about 600 bp in size from the second PCR product (Gel Extraction Kit was purchased from OMEGA) was recovered and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (specific method is the same as above) And screened on LB solid medium containing 50 g/mL ampicillin. Ten white colonies were randomly picked and cultured in LB liquid medium containing 50 g/mL ampicillin, and cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20% by volume, and stored at -80 ° C until use. SEQ ID NO: 6 and the 3' primer AUAP were used for PCR amplification (reaction system and reaction conditions are the same as above), and three positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the gene was obtained. The 5' end of the cDNA.

 After the obtained 5' RACE product was cloned and sequenced, it was spliced with the cloned Th-D178 sequencing result to obtain SEQ ID NO: 17:

 1 AGAAAGGAAG AAGGTTACCT ACCAAATCCC CACACACAAC ACAGCAAAGC ACAGCCTCTC

61 CCTCTCATAT AATCACATGT ATGGGTACCA ACTTGGTCAA AAAGTCACCA CCTTTTATAT

121 TAACCCAAAC TCTCCTCACA TATTGCGTCC TTCTTCTCTT TCTCTCCATA CACACACACA

181 CACATACCAT CTTCTTCACC ATGGGAAGGT CTCCTTGTTG CGAGAAAGCC CACACAAACA 241 AAGGAGCTTG GACCAAAGAA GAAGACCAAC GTCTCGTCGA TTACATCCGT AACCACGGCG

3 01 AAGGTTGCTG GCGTTCTCTT CCCAAATCAG CAGGATTGTT GCGATGTGGT AAAAGTTGTA

361 GACTGAGATG GATTAATTAC CTTCGTCCTG ATCTCAAACG TGGAAACTTC TCTGATGAAG AAGACCAAGT CATCATCAAA CTCCATAGCT TGCTCGGCAA CAAATGGTCA TTGATCGCTG GAAGATTACC AGGAAGAACA GATAATGAAA TCAAAAACTA TTGGAACACT CATATTAAAA GAAAGCTTCT TAGCCATGGA ATCGATCCAC AAACTCATCG TCCGGTTAAA GATTCCGTCG CCGTTACTGA TTCCAAAACG GTTCCGTCTC CGTTAACACA CGCTCACCAA AACGGCGCCG TTGAATACTC TTTCTCCGTT AGACCTAAGA AGGAAATTTC CTCCGATAAC GGCGCTTCCA CAAGCGGCAC GACGACCGAT GAGGATCTCC AGCAGAACGG CGATTGTTAC TACAGTGATG ATTCACGAGA CACAGAGCTG AATCTGGAGT TAACTCTCGG GTCCGGGTCA ACTTCGTGGG TTGGGTCGGA TCGGGTAGTG AGAGCTGGGT TATCGGCAGA TTCGAAGCCG TGGCGGGATC CGGTGACCGC GGTGGTGGCG GCGCGTTTGT CGTTGTTGTA AGAATTGCTA AACGGCAAAA ATCCCCTTTA ACAAAATATC AAAA

 A pair of primers were designed according to the sequence of SEQ ID NO: 17 as follows:

 ThMYBl-2F: SEQ ID NO: 7:

 ATGGGTACCAACTTGGTC

ThMYBl-2R _: SEQ ID NO: 8:

 TTACAACAACGACAAACGCGC

 The full length of ThMYBl-2 was cloned by SEQ ID NO: 7 and SEQ ID NO: 8.

 PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase and cDNA reverse-transcribed cDNA extracted from the leaves of the salt-treated mustard. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 μl, and 30 μΐ double Steamed water. PCR reaction conditions: pre-denaturation at 94 °C for 5 minutes, 33 cycles (denaturation at 94 °C for 30 seconds, annealing at 54 °C for 30 seconds, extension at 72 °C for 2 minutes), extension at 72 °C for 10 minutes.

 PCR amplification product plus A tail: The PCR product was added with 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, de-cleared, dried, and dissolved in 21 μl of double distilled water. Add 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μl 5 ηηΜ dATP, 2.5 μΐ ΙΟχΕχ Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 850 bp was recovered (Omega recovery kit), ligated into pGEM T-easy vector, transformed into JM109 (method as above), and 10 white colonies were randomly picked up in LB liquid containing 50 g/mL ampicillin. The medium was cultured, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20% by volume, and stored at -80 ° C until use. SEQ ID NO: 7 and SEQ ID NO: 8 were subjected to bacterial cell PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the sequence was SEQ ID. NO: 2. According to the nucleotide sequence, the amino acid sequence of the ThMYBl-2 protein encoded by the gene is shown in SEQ ID NO: 1.

Amino acid sequence of MYB 1-2 transcription factor: SEQ ID NO: l 1 MGTNLVKKSP PFILTQTLLT

 21 YCVLLLFLS I HTHTHTIFFT

 41 MGRSPCCEKA HTNKGAWTKE

 61 EDQRLVDYIR NHGEGCWRSL

 81 PKSAGLLRCG KSCRLRWINY

 101 LRPDLKRGNF SDEEDQVI IK

 121 LHSLLGNKWS LIAGRLPGRT

 141 DNEIK YWNT HIKRKLLSHG

 161 IDPQTHRPVK DSVAVTDSKT

 181 VPSPLTHAHQ NGAVEYSFSV

 201 RPKKEISSDN GASTSGTTTD

 221 EDLQQNGDCY YSDDSRDTEL

 241 NLELTLGSGS TSWVGSDRW

 261 RAGLSADSKP WRDPVTAWA

 281 ARLSLL*

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

 1 ATGGGTACCA ACTTGGTCAA AAAGTCACCA CCTTTTATAT TAACCCAAAC TCTCCTCACA

61 TATTGCGTCC TTCTTCTCTT TCTCTCCATA CACACACACA CACATACCAT CTTCTTCACC

121 ATGGGAAGGT CTCCTTGTTG CGAGAAAGCC CACACAAACA AAGGAGCTTG GACCAAAGAA 181 GAAGACCAAC GTCTCGTCGA TTACATCCGT AACCACGGCG AAGGTTGCTG GCGTTCTCTT

241 CCCAAATCAG CAGGATTGTT GCGATGTGGT AAAAGTTGTA GACTGAGATG GATTAATTAC

3 01 CTTCGTCCTG ATCTCAAACG TGGAAACTTC TCTGATGAAG AAGACCAAGT CATCATCAAA

361 CTCCATAGCT TGCTCGGCAA CAAATGGTCA TTGATCGCTG GAAGATTACC AGGAAGAACA

421 GATAATGAAA TCAAAAACTA TTGGAACACT CATATTAAAA GAAAGCTTCT TAGCCATGGA 481 ATCGATCCAC AAACTCATCG TCCGGTTAAA GATTCCGTCG CCGTTACTGA TTCCAAAACG

541 GTTCCGTCTC CGTTAACACA CGCTCACCAA AACGGCGCCG TTGAATACTC TTTCTCCGTT

601 AGACCTAAGA AGGAAATTTC CTCCGATAAC GGCGCTTCCA CAAGCGGCAC GACGACCGAT

661 GAGGATCTCC AGCAGAACGG CGATTGTTAC TACAGTGATG ATTCACGAGA CACAGAGCTG

721 AATCTGGAGT TAACTCTCGG GTCCGGGTCA ACTTCGTGGG TTGGGTCGGA TCGGGTAGTG 781 AGAGCTGGGT TATCGGCAGA TTCGAAGCCG TGGCGGGATC CGGTGACCGC GGTGGTGGCG

841 GCGCGTTTGT CGTTGTTGTA A Example 3 Construction of ThMYBl-2 Gene Plant Expression Vector

 The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter of the ΡΤΠ gene containing the double enhancer was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The inducible promoter rd29A and the terminator Tnos were selected as promoters and terminators of the gene. The construction flow chart is shown in Figure 1.

Using the primers SEQ ID NO: 9 and SEQ ID NO: 10, Pnos was amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and PrimeSTAR HS DNA polymerase of TaKaRa was used. 50 μΐ PCR reaction system: 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ PBI121, 1.0 ^ PrimeSTAR 10 μΜ primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was ligated by EcoRI, Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.

 SEQ ID NO: 9:

 GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 10:

 ATCCAGATC AGATCCGGTGCAGATTATTTG

 Amplification of Tnos with pBI121 as a template using primers SEQ ID NO: 11 and SEQ ID NO: 12

TaKaRa's PrimeSTAR HS DNA Polymerase. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 μl, and 31 μΐ double Steamed water. PCR reaction conditions: pre-variability at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The pCAMBIA2300-2 was obtained by restriction enzyme digestion with pCAMBIA2300-l (Promega T4 ligase kit) by Sacl and EcoRI.

 SEQ ID NO: 11:

 AAGGAGCTCGAATTTCCCCGATCGTTCAAA

SEQ ID NO: 12:

 TCAGAATTCCCAGTGAATTCCCGATCTAGTA

Amplification of Arabidopsis thaliana with primers SEQ ID NO: 13 and SEQ ID NO: 14 using Arabidopsis thaliana (Columbia type, purchased from the Arabidopsis Bioresources Center (www.arabidopsis.org) of the Ohio State University) as a template rd29A promoter (see Zeng J "et L. 2002, Preparation of total DNA from" recalcit rant plant taxa ", Acta Bot Sin, 44 (6):.. 694-697 extraction method Arabidopsis DNA) ₀ using PrimeSTAR HS DNA polymerase from TaKaRa 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ Arabidopsis DNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 13 and SEQ ID NO : 14 each of 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds ), extending at 72 ° C for 10 minutes. The resulting PCR was produced by digestion with HindIII and Pstl. Connected to the object (connection method is the same as above) pCAMBIA2300-2 obtained pCAMBIA2300-3

 SEQ ID NO: 13:

 ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO: 14:

 TGACTGCAGTCCAAAGATTTTTTTCTTTCCAATAG

 ThMYBl-2 was amplified with primers SEQ ID NO: 15 and SEQ ID NO: 16 (template was ThMYB1-2 obtained in Example 2, PrimeSTAR HS DNA polymerase using TaKaRa. 50 μΐ PCR reaction system: 10 ^ 5xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ ThMYBl-2-pGEM, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 15 and SEQ ID NO: 16 each 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: Pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes. After Pstl, Sad digestion, the result The PCR product was ligated (ligation method as above) pCAMBIA2300-3 to obtain the plant expression vector rd29A-ThMYB 1-2-2300.

 SEQ ID NO: 15:

 TGACTGCAGATGGGTACCAACTTGGTC SEQ ID NO: 16:

 AAGGAGCT TTACAACAACGACAAACGCGC Example 4 rd29A-ThMYB 1-2-2300 Expression Vector Transformation Agrobacterium

Agrobacterium tumefaciens LBA4404 (available from Biovector Science Lab, Inc) Preparation of competent cells: Agrobacterium LBA4404 was plated on LB solid medium containing 50 g/ml rifampicin and 50 g/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 μ _§ /ιη1 rifampicin and 50 g/ml streptomycin, and cultured overnight (about 12-16 hours) at 28 °C until the OD600 value was 0.4. , forming a seed bacterial liquid. 5 ml of culture-activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, and cultured at 28 ° C for 2-2.5. Hours to OD _{6 (K)} = 0.8. The ice bath solution was shaken for 10 minutes every 3 minutes to allow the bacteria to enter the dormant state uniformly. Centrifuge at 4000 g for 10 minutes at 4 ° C, discard the supernatant; resuspend the cells by adding 1 ml of ice-cold 10% by volume glycerol, centrifuge at 4000 g for 10 minutes at 4 ° C, collect the precipitate; Cold 10% (volume) glycerin is washed 3-4 times repeatedly; adding appropriate amount of ice pre-cooled 10% (volume) The oil was resuspended in the bacterial pellet to prepare LBA4404 competent cells, which were dispensed at 40 μM/tube and stored at -70 °C until use.

Transformation of Agrobacterium: Melt LBA4404 competent cells on ice, add 1 μΐ of the positive rd29A-ThMYB 1-2-2300 plasmid obtained in Example 3 to 40 μΐ of the competent cells, mix and mix, and then ice bath about 10 minute. Transfer the mixture of competent cells and rd29A-ThMYBl-2-2300 plasmid to a pre-cooled electric shock cup with a micropipette and 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 0.1 cm electric shock cup, the program of the MicroPulser (purchased from Bio-Rad) is set to "Agr" and the shock is applied once. The electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently mix the competent cells with a micropipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 225 rpm for 1 hour at 28 °C. Take 100-200 μL of bacterial solution coated on the corresponding resistant screening medium plate (LB solid medium containing 50 μ _§ /ιη1 rifampicin, 50 μ _§ /ιη1 streptomycin, 50 g/ml card Natamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Agrobacterium-mediated leaf disc transformation was used to obtain transgenic tobacco

Soak the tobacco seeds with 75% alcohol (National Tobacco Medium Term Bank, obtained by the Institute of Tobacco, Chinese Academy of Agricultural Sciences, library number I5A00660) for 30 seconds, wash twice with sterile double distilled water. Soak it in 0.1% liters of mercury for 8 minutes, and wash it twice with sterile 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 H4NO3, 1.5 mM MgS0 ₄ , 3.0 mM CaCl ₂ , 50 μΜ , 100 μΜ Η ₃ ΒΟ ₃ , 100 M MnSO ₄ , 30 M ZnSO ₄ , 1 μΜ Να ₂ Μο0 ₄ , 0.1 M CoCl ₂ , 100 μΜ Να ₂ ΕϋΤΑ, 100 M FeSO ₄ , 7.4 g/L agar, sucrose 30 g/L) , Prepare sterile seedlings. The leaves of the sterile seedlings were cut into 5 mm×5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector rd29A-ThMYB 1-2-2300 in the logarithmic growth phase for 10 minutes to absorb the bacterial liquid in the dark condition. The cells were cultured for 2 days (MS medium). The leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (NAA) + 50 mg/L kanamycin + 500 mg/L cephalosporin). The photoperiod was cultured for about 45 days under the conditions of 10 hours light/14 hours darkness (in which the light intensity was 2000-3000 Lx). After the buds were grown, they were cut and transferred to rooting medium (MS+50 mg/L kanamycin+). The cells were cultured for about 30 days in 500 mg/L cephalosporin. After the root system was developed, the seedlings were transferred to MS medium supplemented with 500 mg/L cephalosporin for number storage. The obtained transgenic tobacco leaves were extracted, and the DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3) using SEQ ID NO: 7 and SEQ ID NO: 8 (50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ DNA, 1.0 μΐ Ex Taq 10 μΜ primer SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 μl, and 35 μΐ double distilled water. PCR reaction conditions: 94 °C pre-denaturation 5 minutes, 33 cycles (denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 2 minutes), extension at 72 °C for 10 minutes), PCR identification, preservation of positive plants for numbering Tod-ToC^ Example 6 Drought tolerance simulation experiment and functional identification of overexpressing ThMYBl-2 transgenic tobacco T _Q The sterilized vermiculite was soaked in 1/2 MS medium. Tod-ToC^ and control tobacco tissue culture seedlings were transplanted to vermiculite, respectively, at 25 °C, 10 hours light culture/14 hours dark culture cycle, 1/2MS every 5 days, 15 days after strong seedling culture, drought Stress test, transgenic tobacco, control tobacco drought for 14 days (without watering), 25 °C, 10 hours light culture / 14 hours dark culture cycle. T _Q transgenic plants (seed T _Q transgenic plants grow into plants) drought resistance identification showed the control plants are wilting serious, and _{T Q C 2, T 0 C} 3 ToC 7, T 0 C 9 T 0 Cio T ₀ Cii T _Q C ₁₄ 29 total seven lines 20 can be tobacco have normal growth, an obvious significant drought tolerance (see FIG. 3, T.C _3, T.C ₉ as an example, T. The results of C ₂ , T.C ₇ , T ₀ Cio T. C _U , T ₀ C ₁₄ are similar to T _Q C ₃ , T _Q C ₉ , not shown here). Example 7 verified at the transcriptional level Γ Λ¾ -2 gene were taken control tobacco, no significant drought tolerant transgenic tobacco T _Q generation plants significantly drought tolerant transgenic tobacco T _Q-generation plants (grow well) arid 14 days leaves 0.05 g, Total RNA was extracted using a 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 respective RNA concentrations. Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay LIBOX Superscript III Reverse Transcriptase (.2\ag total RNA as a template, reverse transcription primer SEQ ID NO: 8). The relative expression of MYB 1 -2 protein was detected by amplifying ThMYB 1 -2 by SEQ ID NO: 7 and SEQ ID NO: 8. The PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 μl, and 30 μΐ double Steamed water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 29 cycles (denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes. The product electrophoresis results are shown in Figure 4: M is DNALadder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-4 for control tobacco, 5-20 for significantly drought-tolerant transgenic tobacco T _Q plants, 21-24 for not significantly drought-tolerant transgenic tobacco T _Q plants . The strip size shown in the figure is consistent with the size of ThMYBl-2 (about 900 bp). The results show that the control tobacco no transcript was detected signal exogenous gene ThMYBl-2 is significantly drought transfected gene transcription tobacco T _Q progenies exogenous gene ThMYBl-2 is strong, no significant drought tolerant transgenic tobacco T _Q progenies exogenous gene ThMYBl-2 transcription is weak or not transcribed.

Claims

Claim A MYB-like transcription factor of salt mustard, the sequence of which is shown in SEQ ID NO: 1.  2. A gene encoding the MYB-like transcription factor of claim 1, the sequence of which is SEQ ID NO: 2. A recombinant expression vector obtained by inserting the gene of claim 2 into an expression vector, and a nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector, Preferably, the expression vector is pCAMBIA2300.  4. The recombinant expression vector of claim 3 which is the rd29A-ThMYB 1-2-2300 vector shown in Figure 2.  A recombinant cell comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4; preferably, the recombinant cell is a recombinant Agrobacterium cell.  A method for improving drought tolerance of a plant, comprising: introducing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 into a plant or plant tissue and expressing the gene; preferably, the plant It is tobacco.  A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions effective to produce a plant.  8. The method of claim 7 wherein the plant is tobacco.  The gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 5 for use in improving drought tolerance of a plant and for use in plant breeding.  10. The use of claim 9, wherein the plant is tobacco.