WO2015042738A1

WO2015042738A1 - Thellungiella halophila homeotic-leucine zipper protein hdbzip-3, coding gene of same, and application thereof

Info

Publication number: WO2015042738A1
Application number: PCT/CN2013/001157
Authority: WO
Inventors: 陈文华; 孙超; 崔洪志
Original assignee: 创世纪转基因技术有限公司
Priority date: 2013-09-26
Filing date: 2013-09-26
Publication date: 2015-04-02
Also published as: CN105452278A

Abstract

Provided are a Thellungiella halophila-sourced leucine zipper protein HDbZIP-3, a coding gene of same, and an application thereof in cultivating a transgenic plant of increased drought tolerance.

Description

A small salt mustard homeotype-leucine zipper protein HDbZHV? and its coding gene and application

FIELD OF THE INVENTION The present invention relates to plant proteins and their coding genes and applications, and more particularly to a homeobox-leucine zipper protein HDbZIP-3 derived from Brassica napus and its coding gene, and its improvement in drought tolerance Application in transgenic plants. BACKGROUND OF THE INVENTION Stresses such as temperature, salting and drought can cause serious damage to the growth and development of higher plants, resulting in reduced crop yields, degraded quality, and serious threats to agricultural production and the natural environment. Among them, the impact of drought on crop yields ranks first in many natural adversities, and its harm is equivalent to the sum of other disasters. Many regions are the bottleneck of agricultural development. According to statistics, the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid regions account for 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares. Cubic meters, due to lack of water, less than 350-400 billion kilograms of grain; 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.

Most of the drought tolerance of plants belongs to the quantitative traits controlled by multiple genes. The use of conventional breeding methods to improve the drought resistance of crops is limited by the long cycle and lack of excellent germplasm resources. Recent studies on transcriptomics, proteomics and gene expression regulation have revealed the molecular mechanism of plant drought stress. At present, the use of drought stress-related genes to improve the drought resistance of plants has become a research hotspot of plant resistance to molecular biology and an important research direction of plant stress resistance genetic engineering.

When plants are stressed by stress, they will respond accordingly to reduce or eliminate the damage caused by stress. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. 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 have yet 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 study of the entire signaling network system. SUMMARY OF THE INVENTION The present inventors used SSH (Suppression Subtractive Hybridization) in combination with RACE (Rapid Amplification of cDNA Ends) The gene encoding a leucine zipper protein of small salt mustard (herein named HDbZIP-3) was cloned and its DNA sequence was determined. Moreover, it was found that after being introduced into plants for over-expression, the drought tolerance of the 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 homeotype-leucine zipper protein HDbZIP-3 of the small salt mustard (herein named ThHDbZIP-3; preferably, the sequence thereof is 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 a basic vector for constructing the recombinant expression vector, And the nucleotide sequence of the gene is operably linked to the expression control sequence of the base vector; preferably, the base vector is pCAMBIA2300; preferably, the recombinant expression vector is 35S- shown in Figure 2 73⁄4HZ1⁄2Z/P-3-2300 carrier.

The 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 Arabidopsis thaliana.

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 or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Tissue; Preferably, the plant is Arabidopsis thaliana.

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 Arabidopsis thaliana.

The seventh aspect of the present invention provides the gene-encoded protein according to the first aspect of the present invention, which has an amino acid sequence as shown in SEQ ID NO: 1. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (358-7Μ/Ζ3⁄4Ζ/Ρ-3-2300;) of ThHDbZIP-3 (Fig. la-lb). Figure 2 is a plasmid map of the plant expression vector of ThHDbZIP-3 (358-7Μ/Ζ1⁄2Ζ/Ρ-3-2300;).

Figure 3 shows the results of drought tolerance simulation experiments of 73⁄4HZ1⁄2Z/P-3 T1 transgenic Arabidopsis plants (T106 in the figure) and non-transgenic Arabidopsis plants (in the figure, CK) as controls. (Fig. 3a is an Arabidopsis plant that is normally grown for 20 days; Fig. 3b is an Arabidopsis plant that has been subjected to drought treatment for 14 days after normal growth for 20 days). Fig. 4 Results of changes in ABA content of T1 transgenic Arabidopsis plants and control plants under drought stress and normal growth conditions. 1-7 is a strain (from left to right): T101, Τ102, Τ103, Τ104, Τ105, Τ106, CK, wherein Τ101, Τ102, Τ103, Τ104, Τ105, T106 are transgenic plants, and CK is a control plant.

Figure 5 shows the results of protein expression verification at the transcriptional level of transgenic T1 Arabidopsis plants and non-transgenic control plants. M is DNA Ladder Marker (DL2000, TakaRa), 1-6 is a drought-tolerant transgenic Arabidopsis T1 plant (in order: Τ101, Τ102, Τ103, Τ104, Τ105, Τ106), 7-11 is a drought-tolerant transgenic Arabidopsis thaliana 1st generation plants, 12-16 are non-transgenic Arabidopsis controls. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.

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

The specific method is:

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

(1) Test materials:

Small salt mustard (T ellungiella halophila, purchased from the Yanlan Plant Breeding Center of Ulan Buh and Desert Green Botanical Garden, Bayannaoer City, Inner Mongolia, China), seeded on sterilized vermiculite, at 25 ° C, photoperiod 16 hours light / Incubate in 8 hours dark (light intensity 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 NH ₄ N0 ₃ , 0.75 mM MgS0 ₄ , 1.5 mM CaCl ₂ , 50 μΜ KI, 100 μΜ Η ₃ ΒΟ ₃ , 100 M MnSO ₄ , 30 μΜ ZnS0 ₄ , 1 μΜ Να ₂ Μο0 ₄ , 0.1 μΜ CoCl ₂ , 100 μΜ Na ₂ EDTA, 100 M FeSO ₄ ). It was used for experiments when the seedlings were as high as 25-30 cm.

(2) Material handling:

The test seedlings were divided into 2 groups, 4 pots per group and 1 pot per pot. The first group was a control group, cultured at 25 ° C, photoperiod of 16 hours light / 8 hours dark, and normal watering. The second group is the drought treatment group, 25 °C, light The cells were cultured under the conditions of 16 hours light/8 hours dark, stopped watering, and treated for 10 days. After the treatment, the leaves of the top 1/3 of the seedlings of the two groups were cut out in time, quickly frozen with liquid nitrogen, and stored in a refrigerator at -70 °C. .

(3) Total RNA extraction:

The leaves of the small salt mustard leaves of the control group and the drought treatment group were respectively 0.5 g, and the total RNA of the leaves of the small salt mustard was extracted with the plant RNA extraction kit (purchased from invitrogen). The absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001. The OD ₂₆₀ / OD ₂₈₀ ratio was 1.8-2.0, indicating a higher total RNA purity; 1.0% agarose gel The total RNA was detected by electrophoresis, and the brightness of the 28S band was about twice that of the 18S band, indicating good RNA integrity. mRNA was isolated using Qiagen's purification of poly A+ RNA from total RNA.

(4) Suppression of subtractive hybridization:

Suppression Subtractive Hybridization performed by PCR-select ^TM cDNA Clontech's method shown Subtraction Kit kit. The Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and then subtracted hybridization using 2 Tester cDNA and P 2 g 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. The two first subtractive hybridization products were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and then the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.

In order to increase the validity of the Expressed sequence tag (EST) (Unigene), avoid the gene-free cleavage site and the obtained sequence in the untranslated region, this experiment simultaneously uses the endonuclease Haelll to tester cDNA according to the above steps. The cDNA was digested with Driver cDNA and subjected to two forward subtractive hybridizations and two inhibitory PCR amplifications. Finally, the second inhibitory PCR products of the two groups of forward subtractive hybridization cDNA fragments were combined.

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

The second PCR product of the combined forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM-T Easy (purchased from Promega kit) according to the procedure of the pGEM-T Easy kit The vector is ligated, and the specific steps are as follows: The following components are sequentially added by using a 200 μΐ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 μ1, 2χΤ4 ligase buffer 5 , pGEM-T Easy vector 1 11, T4 DNA ligase 1 μΐ, ligated overnight at 4 °C. Take 10 ligation reaction products, add to 100 competent E. coli JM109 (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, ice bath for 2 minutes, and add 250 μL of LB medium (1% Tryptone from OXOID) , 0.5% Yeast Extract was purchased from OXOID, 1% NaCl purchased from Sinopharm) was placed in a 37 ° C water bath, shaken at 225 rpm for 30 minutes, and 200 μL of bacterial solution was applied to 50 g/mL ampicillin (purchased from Tiangen Biochemical Technology (Beijing). ))) LB (ibid.) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA, lg packaged into 20 mg/ml mother liquor, working concentration: 200 μΙ/100 ml LB medium above IPTG was purchased from TaKaRa, and 5 g was packaged into a mother liquor at a concentration of 100 mM. The working concentration was: 100 μΙ/100 ml of LB medium above the mother liquor. The cells were incubated at 37 ° C for 18 hours on a solid culture plate. Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 198 white colonies (number: Gh-B001 to Gh-B198). All white clones were inoculated separately in 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 g/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-selectTM cDNA Subtraction Kit kit) were used to verify the PCR amplification of the bacteria, and 166 positive clones were obtained, and then all the positive clones were sent to the UK. Base (Shanghai) Trading Co., Ltd. sequencing.

(6) cDNA sequencing analysis of differential clones:

After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 123 ESTs (Unigene;) were obtained. After analysis, there are 22 contigs with 101 single sequences. It was found by BlastN that 53 ESTs (Unigene) have homologous sequences in GenBank, 21 EST functions are unknown or hypothetical proteins, and another 27 have not obtained homologous matches, presumably at the 3' or 5' end. A shorter sequence of regions. Example 2 Salt mustard leucine zipper protein encoding gene ThHDbZIP-3 clone

After the cloned YLS-120 was removed from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to the leucine zipper protein. In this paper, the full-length coding gene corresponding to the clone YLS-120 was named as ThHDbZIP-3, its corresponding protein is named HDbZIP-3.

SEQ ID No: 3

1 TTTGGAGCTG AACG TGGAT CGCTACTCTC CAAAGAATGT GCGAGAGATT CAAGACTGTA

61 CTAGAACCCG CAACATCTTC CCGCGATCTC AAAGGAGTGA TTCCATCTCT GGAAGGGAAG 121 AGAAGTATAA TGAGACTATC TCAAAGAATG GTAAGCAACT TTGC TGAG CGT GGCACA

181 TCTAACAACA ATGGCTCAAC GGTTATTTCC GGGTTGGAGG AGTTTGGAAT CCGTGTGACC

241 TCGTATAAGA GTCAGCATGA ACCAAATGGA ATGGTTCTAT GTGCAGCCAC TAGTTTCTGG

301 CTCCCAATTT CTCCACAAAA CGTCTTCAAT TTCCTCAAAG ATGAAAGAAC TCGGCCACAG

361 GGGACGTTC TTTCGAATGG AAATTCTGTT CAAGAAGTTG CTCATATCGC AAACGGCTCA 421 CACCCTGGAA ACTGCATTTC GGT CTCCGC GGATTCAA G CATCTTCATC ACAAAACAAC

481 ATGCTGATTC TACAAGAAAG CTGGGTAGAC TCATCAGGCT CGCTTGTGGT GTACACTCCG

541 GTGGATCTAC CAGCACTGAA CATGGCAATG AGCGGTCAAG ACACATCTTA TGTTCCCATT

601 CTACCCTCGG GT TCGCCGT TTCACCAGAC GGAACCAGGA ATAACCAGCT CGCAGAGCAC

661 AAAGTTGAAG GAGGAGGAGG TTCATTGATA ACGGT GGTT TTCAGATAAT GGTGAGTAGT 721 TTGCAGTCAG CGAAAT GAA TGTGGAGTCA ATGGAAACAG TTAATAATCT CATCAGCACC 781 ACTGTCCACC AAATTAAAAC CACCTTGAAC TGTCCTTCAA CTGCTTGA HDbZIP-3 full-length coding gene clone

According to the sequence analysis of SEQ ID No: 3 which has been obtained, SEQ ID No: 3 is the 3-terminal sequence of the coding gene HZ1⁄2Z/P-3. Based on the sequence of SEQ ID NO: 3 which has been obtained, the following three specific primers were designed as specific primers for reverse transcription primers and 5 'RACE.

YLS-120GSP 1 ( SEQ ID NO: 4 ) :

TGAGCCGTTTGCGATATGAG YLS-120GSP2 ( SEQ ID NO: 5 ) :

CCATTCGAAAGAACGTCCCA

YLS-120 GSP3 ( SEQ ID NO: 6 ) :

TTTGGAGCTGAACGTTGGAT

The kit comes with universal primers:

AAP ( SEQ ID NO: 7 ) :

GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG

AUAP ( SEQ ID NO: 8 ) :

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

Using YLS-120GSP1 (SEQ ID NO: 4) as a reverse transcription primer, reverse transcription using small salt mustard mRNA as a template to obtain a cDNA template, and then adding a Poly C tail according to the procedure in the above 5' RACE kit instructions. The first round of PCR amplification was carried out using the product after tailing as a template. The primer used was SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, I is a hypoxanthine modified a, c, g or t), 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 (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 4 and SEQ ID NO: 7 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 °C for 50 seconds, 58 °C for 50 seconds, 72 °C for 90 seconds), 72 °C for 10 minutes.

The obtained PCR product was diluted 50-fold 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: 5 and the universal primer SEQ ID NO: 8. 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 primers SEQ ID NO: 5 and SEQ ID NO: 8 each 2.0 μl, and

35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation for 50 seconds at 94 °C, annealing for 50 seconds at 58 °C, extension of 90 seconds at 72 °C), extension at 72 °C for 10 minutes. A strip of about l. lKbp in the second PCR product was recovered (Gel Extraction Kit was purchased from OMEGA), and it was ligated to pGEM-T Easy Vector, and then converted to JM109 (the specific method is the same as above), and randomly picked 6 One white colony was inoculated separately in LB liquid medium containing 50 g/mL ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20% (v/v), and stored at -80 ° C until use. The primers SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were used for PCR amplification (reaction system and reaction conditions as above), and 6 positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing, a 5' end sequence of the cDNA of the gene was obtained.

The obtained 5' RACE product clone YL16-3 was sequenced to obtain the sequence of SEQ ID NO: 9:

GGGGGGGGGG AACTTGGTTT TATCAGACAT AGATAAGTCT CTTA GACCA GCATCGCTGT

61 GACCGCTATC GAAGAATTGC TTAGGCTCAC ACAAACAAAT GAGCCTCTAT GGATCAAAAC

121 GGATGGATGC AGAGAAATTC TCAATCTCGA AAGCTATGAG AATATATTCT CAAGATCAAG

181 TAGCCG GGA GGAAAGAACC ATAACC TCG AAAGGAAGCA TCTAGATTTT CTGGGGTTGT

241 TTTCACIAAT GCTATGACCC TTGTTGATAT GCTCATGGAC TCGGTCAAAT CTGCAGAGCT

301 T TTCCCTCA ATTGTTGCAA CATCTAAAAC CCTTGCAG G ATTTCATCCA GCTTGCGCGG

361 AAACCG GCA GA GCATTGC ATTTGATGCT TGAAGAGCTT CAAGTGCITT CCCCATTGGT

421 ACCGACGCGT GAGTTCTGGG GGCIAAGATA TTGTCAGCAA ATACAACATG GAACTTGGGC

481 AATTGTAAAT GTCTCCTA G AGTTCCCTCA GTTTATATCT CAT CTCGGT CGTATCGGTA

541 TCCTTCTGGT TGCTTGATTC AAGACATGTC CAATGGCTAT TCCAAGGTTA T TGGGTCGA

601 ACATGTTGAT ACGGAGGAGC AAGAAGCGGT TCACGAGA G TTTAAAGATA ATGTCCGTCA

661 AGGATTAGCT TTTGGAGCTG AACGTTGGAT CGCTACTCTC CAAAGAATGT GCGAGAGATT

721 CAAGACTGTA CTAGAACCGG CAACATCTTC CCGCGATCTC AAAGGAGTGA TTCCATCTCT

781 GGAAGGGAAG AGAAGTATAA TGAGACTATC TCAAAGAA G GTAAGCAACT TTGCTTGAG

841 GGTTGGCACA TCIAACAACA ATCGCTCAAC GGTTATTTCC GGGT GGACG AGTTTGGAAT

901 CCGTGTGACC TCGTATAAGA GTCAGCATGA ACCAAATGGA ATGGTTCTAT GTGCAGCCAC

961 TAGTTTCTGG CTCCCAATTT CTCCACAAAA CGTC TCAAT TTCCTCAAAG ATGAAAGAAC

1021 TGGGCCACAG TGGGACGT C T TCGAATGG The sequence SEQ ID NO: 9 obtained by 5 ' RACE was spliced with the obtained sequence SEQ ID NO: 3 to obtain SEQ ID NO: 10:

1 GGGGGGGGGG AACTTGGTTT TATCAGACAT AGATAAGTCT CTTA GACCA GCATCGCTGT

61 GACCGCTATC GAAGAATTGC TTAGGCTCAC ACAAACAAAT GAGCCTCTAT GGATCAAAAC

121 GGATGGATGC AGAGAAATTC TCAATCTCGA AAGCTATGAG AATATATTCT CAAGATCAAG 181 TAGCCG GGA GGAAAGAACC ATAACC TCG AAAGGAAGCA TCTAGATTTT CTGGGGTTGT

241 TTTCACIAAT GCTATGACCC TTGTTGATAT GCTCATGGAC TCGGTCAAAT CTGCAGAGCT

301 T TTCCCTCA ATTGTTGCAA CATCTAAAAC CCTTGCAG G ATTTCATCCA GCTTGCGCGG

361 AAACCG GCA GA GCATTGC ATTTGATGCT TGAAGAGCTT CAAGTGCITT CCCCATTGGT

421 ACCGACGCGT GAGTTCTGGG GGCIAAGATA TTGTCAGCAA ATACAACATG GAACTTGGGC 481 AATTGTAAAT GTCTCCTATG AGTTCCCTCA GTTTATATCT CAT CTCGGT CGTATCGGTA

541 TCCTTCTGGT TGCTTGATTC AAGACATGTC CAATGGCTAT TCCAAGGTTA T TGGGTCGA

601 ACATGTTGAT ACGGAGGAGC AAGAAGCGGT TCACGAGA G TTTAAAGATA ATGTCCGTCA

661 AGGATTAGCT TTTGGAGCTG AACGTTGGAT CGCTACTCTC CAAAGAATGT GCGAGAGATT 721 CAAGACTGTA CTAGAACCGG CAACATCTTC CCGCGATCTC TTCCATCTCT

781 GGAAGGGAAG AGAAGTATAA TGAGACTATC TCAAAGAA G TTGCTTGAG

841 GGTTGGCACA TCIAACAACA ATCGCTCAAC GGTTATTTCC AGTTTGGAAT

901 CCGTGTGACC TCGTATAAGA GTCAGCATGA ACCAAATGGA GTGCAGCCAC

961 TAGTTTCTGG CTCCCAATTT CTCCACAAAA CGTC TCAAT ATGAAAGAAC

1021 TGGGCCACAG TGGGACGT C T TCGAATGG AAATTCTGTT CTCATATCGC

1081 AAACGGCTCA CACCCTGGAA ACTGCATTTC GGT CTCCGC CATCTTCATC

1141 ACAAAACAAC ATGCTGATTC TACAAGAAAG CTGCGTAGAC CGCTTGTGGT

1201 GTACACTCGG GTGGATCTAC CAGCACTGAA CATGGCAA G ACACATCTTA

1261 TGTTCCCATT CTACCCTCGG GT TGGCCGT TTCACCAGAC ATAACCAGCT

1321 CGCAGAGCAC AAAGTTGAAG GAGGAGGAGG TTCATTGATA TTCAGATAAT

1381 GGTGAGTAGT TTGCAGTCAG CGAAATTGAA TGTGGAGTCA TTAATAATCT

1441 CATCAGCACC ACTGTCCACC AAATIAAAAC CACCTTGAAC CTGCTTGA According to the sequence search analysis of SEQ ID NO: 10, SEQ ID NO: 10 is not the full length sequence of Γ /Ζ1⁄2Ζ/Ρ-3. Further 5' RACE is required to obtain the full length of the gene. According to the sequence of SEQ ID NO: 10, the following three specific primers were designed as specific primers for reverse transcription primers and 5' RACE.

YLS-120GSP1 : SEQ ID NO: 11 :

GCTCTGCAGATTTGACCGAG

Abdomen 3⁄4 AAS i

YLS-120GSP2: SEQ ID NO: 12:

GCATATCAACAAGGGTCATAGC ϋ=

YLS-120 GSP3 : SEQ ID NO: 13 :

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

Using YLS-120GSP1 (SEQ ID NO: 11) as a reverse transcription primer, reverse transcription using small salt mustard mRNA as a template to obtain a cDNA template, and then adding a Poly C tail according to the procedure in the above 5' RACE kit instructions. The first round of PCR amplification was carried out using the tailed product as a template. The primers used were SEQ ID NO: 11 and the universal primer SEQ ID NO: 7 (the kit is self-contained, and I is hypoxanthine modified a, c, g or t), 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 (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 7 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation for 50 seconds at 94 ° C, annealing for 50 seconds at 56 ° C, extension of 90 ° C for 90 seconds), extension at 72 ° C for 10 minutes.

The resulting PCR product was diluted 50-fold with double distilled water and 2.0 μL was used as a template, and a second round of PCR amplification was carried out using SEQ ID NO: 12 and the universal primer SEQ ID NO: 8, 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 primers SEQ ID NO: 12 and SEQ ID NO: 8 each 2.0 μl, and

35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 minutes, 33 cycles (denaturation for 50 seconds at 94 °C, 50 seconds for 50 seconds, extension for 90 seconds at 72 °C), and extension of 72 V for 10 minutes. A strip of approximately l.lKbp in the second PCR product was recovered (Gel Extraction Kit was purchased from OMEGA) and ligated into pGEM-T Easy Vector, then converted to JM109 (specific method as above), randomly picked 6 One white colony was inoculated separately in LB liquid medium containing 50 μl/ηιΣ ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20% (v/v), and stored at -80 ° C until use. The primers SEQ ID NO: 12 and the 3 '-end primer SEQ ID NO: 13 were used for PCR amplification (reaction system and reaction conditions as above), and 6 positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing, a 5' end sequence of the cDNA of the gene was obtained.

The resulting 5 'RACE product clone was sequenced to obtain the sequence of SEQ ID NO: 14:

1 GGGGGGGGGG ATGGAGTTTC TGGGCGACAG CCAAAATCAC GACAGTTCTG AAATGGACAA

61 GAGGAAGAAG AAGAAGCGAT T CACCGCCA CACACCTCAC CAGATCCAAC GCCTGGAATC

121 AACTTTCAAT GAGTGTCAAC ATCCAGATGA GAAACAGAGG TGCAACTTA GCAGAGAATT

181 GGGTTTAGCT CCAAGACAGA TCAAGTTTTG GTTTCAGAAC AGAAGAACAC AAAAGAAGGC 241 ACAACAGGAG AGAGCTGATA ACTGTGCACT CAAGGAAGAG AATGATAAGA TCGT GCGA

301 AAACATTGCG ATTAGAGAAG CTATAAAACA CGCCAT TGC CCTAACTGTG GTGATTCTCC

361 GTTAA GAA GACTCTTACT TTGATGAACA GAAGCTTCGA ATCGAAAATG CACAGCTAAG

421 ACAAGAGCTT GAAAGGGTTT CAAGCATTGC AGCTAAATTC ATAGGACCTC TGCTCATCT

481 CCCACCAATA CTAAATCCGA TGCACATTTC GCCATCAGAG TTATTCCATA GTGGTCCTTC 541 GCTTGATTTT GATCTTCTTC CAGGAAGTTG TTCTTCAA G GCTACTGTTC CTAATTTACC

601 ATCTCAGCCA AACTTGGTTT TATCAGACAT AGATAAGTCT CTTATGACCA GCATGGCTGT

661 GACCGCTATC GAAGAATTGC TTAGGCTCAC ACAAACAAAT GAGCCTCTAT GGATCAAAAC

721 GGATGGATGC AGAGAAATTC TCAATCTCGA AAGCTATGAG AATATATTCT CAAGATCAAG

781 TAGCCG GGA GGAAAGAACC ATAACC TCG AAAGGAAGCA TCTAGATTTT CTGGGGTTGT 841 TTTCACIAAT GCTATGACCC TTGTTGATAT GC The sequence of SEQ ID NO: 14, obtained by 5 'RACE, was spliced with the obtained splicing sequence SEQ ID NO: 10 to obtain SEQ ID NO: 15:

1 GGGGGGGGGG ATGGAGTTTC TGGGCGACAG CCAAAATCAC GACAGTTCTG AAATGGACAA 61 GAGGAAGAAG AAGAAGCGAT T CACCGCCA CACACCTCAC CAGATCCAAC GCCTGGAATC

121 AACTTTCAAT GAGTGTCAAC ATCCAGATGA GAAACAGAGG TTGCAACTTA GCAGAGAATT

181 GGGTTTAGCT CCAAGACAGA TCAAGTTTTG GTTTCAGAAC AGAAGAACAC AAAAGAAGGC

241 ACAACAGGAG AGAGCTGATA ACTGTGCACT CAAGGAAGAG AATGATAAGA TCGT GCGA

301 AAACATTGCG ATTAGAGAAG CTATAAAACA CGCCAT TGC CCTAACTGTG GTGATTCTCC 361 GTTAA GAA GACTCTTACT TTGATGAACA GAAGCTTCGA ATCGAAAATG CACAGCTAAG

421 ACAAGAGCTT GAAAGGGTTT CAAGCATTGC AGCTAAATTC ATAGGACCTC TGCTCATCT

481 CCCACCAATA CTAAATCCGA TGCACATTTC GCCATCAGAG TTATTCCATA GTGGTCCTTC

541 GCTTGATTTT GATCTTCTTC CAGGAAGTTG TTCTTCAATG GCTACTGTTC CTAATTTACC

601 ATCTCAGCCA AACTTGGTTT TATCAGACAT AGATAAGTCT CTTATGACCA GCATGGCTGT 661 GACCGCTATC GAAGAATTGC TTAGGCTCAC ACAAACAAAT GAGCCTCTAT GGATCAAAAC

721 GGATGGATGC AGAGAAATTC TCAATCTCGA AAGCTATGAG AATATATTCT CAAGATCAAG

781 TAGCCG GGA GGAAAGAACC ATAACC TCG AAAGGAAGCA TCTAGATTTT CTGGGGTTGT

841 TTTCACIAAT GCTATGACCC TTGTTGATAT GCTCATGGAC TCGGTCAAAT CTGCAGAGCT 901 T TTCCCTCA ATTGTTGCAA CATCTAAAAC CCTTGCAG G ATTTCATCCA GCTTGCGCGG

961 AAACCG GCA GA GCATTGC ATTTGATGCT TGAAGAGCTT CAAGTGCITT CCCCATTGGT

1021 ACCGACGCGT GAGTTCTGCG GGCTAAGATA TTGTCAGCAA ATACAACATG GAACTTGGGC

1081 AATTGTAAAT GTCTCCTATG AGTTCCCTCA GTTTATATCT CAT CTCGGT CGTATCGGTA

1141 TCCTTCTGGT TGCTTGATTC AAGACATGTC CAATGGCTAT TCCAAGGTTA T TGGGTCGA

1201 ACATGTTGAT ACGGAGGAGC AAGAAGCGGT TCACGAGA G TTTAAAGATA ATGTCCGTCA

1261 AGGATTAGCT TTTGGAGCTG AACGT GGAT CGCTACTCTC CAAAGAATGT GCGAGAGATT

1321 CAAGACTGTA CTAGAACCCG CAACATCTTC CCGCGATCTC AAAGGAGTGA TTCCATCTCT

1381 GGAAGGGAAG AGAAGTATAA TGAGACTATC TCAAAGAA G GTAAGCAACT TTGCTTGAG

1441 CGTTGGCACA TCTAACAACA ATCGCTCAAC GGTTATTTCC GGGT GGACG AGTTTGGAAT

1501 CGGTGTGACC TCGTATAAGA GTCAGCATGA ACCAAATGGA ATGGTTCTAT GTGCAGCCAC

1561 TAGTTTCTGG CTCCCAATTT CTCCACAAAA CGTC TCAAT TTCCTCAAAG ATGAAAGAAC

1621 TGGGCCACAG TGGGACGT C T TCGAATGG AAATTCTGTT CAAGAAGTTG CTCATATCGC

1681 AAACGGCTCA CACCCTGGAA ACTGCATTTC GGT CTCCGC GGATTCAATG CATCTTCATC

1741 ACAAAACAAC ATGCTGATTC TACAAGAAAG CTGCGTAGAC TCATCAGGCT CGCTTGTGGT

1801 GTACACTCGG GTGGATCTAC CAGCACTGAA CATGGCAA G AGCGGTCAAG ACACATCTTA

1861 TGTTCCCATT CTACCCTCGG GT TGGCCGT TTCACCAGAC GGAACCAGGA ATAACCAGCT

1921 CGCAGAGCAC AAAGTTGAAG GAGGAGGAGG TTCATTGATA ACGGTTGG T TTCAGATAAT

1981 GGTGAGTAGT TTGCAGTCAG CGAAAT GAA TGTGGAGTCA ATGGAAACAG TTAATAATCT

2041 CATCAGCACC ACTGTCCACC AAATIAAAAC CACCTTGAAC TGTCCTTCAA CTGCTTGA According to the sequence search analysis of SEQ ID NO: 15, SEQ ID NO: 15 is: the full length sequence of ^^^^/^-^. A pair of primers were designed according to the sequence of SEQ ID NO: 15 as follows: ThHDbZIP-3F (SEQ ID NO: 16): ATGGAGTTTCTCGGCGACAG

ThHDbZIP-3R (SEQ ID NO: 17): TCAAGCAGTTGAAGGACAGTTC AP (SEQ ID NO: 18):

GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT The ThHDbZIP-3 full-length coding sequence was cloned by SEQ ID NO: 16 and SEQ ID NO: 17.

The small salt mustard RNA was extracted, and the primer SEQ ID NO: 18 was used as the reverse transcription primer to obtain the cDNA of the small salt mustard. The PCR reaction was carried out using the cDNA of the small salt mustard using the PfuUltra II Fusion HS DNA Polymerase of Stratagene. 50 μΐ PCR reaction system: 5 μΐ lO PfuUltra II reaction Buffer, 0.5 μΐ 25 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 11, and 37.5 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation at 95 °C for 25 seconds, annealing at 58 °C for 25 seconds, extension at 72 °C for 1 minute), extension at 72 °C for 5 minutes. PCR amplification product plus A tail: PCR product hydration to 400 μ1, first remove the protein with chloroform once, add the supernatant to add 3 Μ sodium acetate solution 40 μl, add 2 times of absolute ethanol, -20 ° C for 10 minutes, Centrifuge, remove the supernatant, allow to dry, and dissolve in 21 μl of double distilled water. Add 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μΐ 5 mM dATP, 1.0 μΐ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 2.1 Kbp was recovered (Omega recovery kit), ligated into pGEM T-easy vector (73⁄4HZ1⁄2Z/P-3-pGEM plasmid), then transformed into JM109, and 6 white colonies were randomly picked and inoculated separately. Incubate in LB liquid medium containing 50 g/mL ampicillin, incubate at 37 °C overnight, add glycerol to a final concentration of 20% (v/v), and store at -80 °C until use. The primers SEQ ID NO: 16 and SEQ ID NO: 17 were used for PCR amplification (reaction system and reaction conditions as above), and 6 positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. SEQ ID NO: 2, the amino acid sequence of the encoded protein is SEQ ID NO: 1

Amino acid sequence of HDbZIP-3 protein: SEQ ID NO: 1

1 MEFLGDSQNH DSSE DKRKK KKRFHRHTPH QIQRLESTFN ECQHPDEKQR LQLSRELGLA

61 PRQIKFWFQN RRTQKKAQHE RADNCALKEE NDKIRCENIA IREAIKHAIC PNCGDSPVME

121 DSYFDEQKLR IENAQLRQEL ERVSSIAAKF IGPLAHLPPI LNPMHISPSE LFHSGPSLDF 181 DLLPGSCSSM ATVPNLPSQP NLVLSDIDKS LM SIAVTA EELLRLTQTN EPLWIK DGC

241 REIL LESYE NIFSRSSSRG GKNHNLRKEA SRFSGWFTN AMTLVDMLMD SV SAELFPS

301 IVATSK LAV ISSSLRG RA DALHLMLEEL QVLSPLVP R EFCGLRYCQQ IQHGTWAIV

361 VSYEFPQFIS HSRSY YPSG CLIQDMSNGY SKVIWVEHVD TEEQEPVHE FKDNVRQGLA

421 PGAERWIATL QR CERFK L LEPATSSRDL KGVIPSLEGK RSIMRLSQR VSNFCLSVGT 481 SNNNRSTVIS GLDEFGIRVT SY SQHEPNG MVLCAATSFW LPISPQNVFN FLKDERTRPQ

541 WDVLSNGNSV QEVAHIA GS HPGNCISVLR GFNASSSQ N MLILQESCVD SSGSLWYTP

601 VDLPAL MAM SGQDTSYVPI LPSGFAVSPD GTRNNQLAEH KVEGGGGSLI TVGFQIMVSS

661 LQSAKL VES METVNNLIST TVHQIK TLN CPSTA

Nucleotide sequence of the ThHDbZIP-3 encoding gene: SEQ ID NO:

ATGGAGTTTC TGGGCGACAG CCAAAATCAC GACAGTTCTG AAATGGACAA GAGGAAGAAG

61 AAGAAGCGAT T CACCGCCA CACACCTCAC CAGATCCAAC GCCTGGAATC AACTTTCAAT

121 GAGTGTCAAC ATCCAGATGA GAAACAGAGG TTGCAACTTA GCAGAGAATT GGGTTTAGCT

181 CCAAGACAGA TCAAGTITTG GTTTCAGAAC AGAAGAACAC AAAAGAAGGC ACAACACGAG

241 AGAGCTGATA ACTG GCACT CAAGGAAGAG AATGATAAGA TCGT GCGA AAACATTGCG

301 ATTAGAGAAG CTATAAAACA CGCCAT TGC CCTAACTGTG GTGATTCTCC TGTTAATGAA

361 GACTCTTACT TTGA GAACA GAAGCTTCGA ATCGAAAATG CACAGCTAAG ACAAGAGCTT

421 GAAAGGGTTT CAAGCATTGC AGCTAAATTC ATAGGACCTC TTGCTCATCT CCCACCAATA

481 CTAAATCCGA TGCACATTTC GCCATCAGAG TTATTCCATA GTGGTCCTTC GCTTGATTTT

541 GATCTTCTTC CAGGAAGTTG TTCTTCAATG GCTACTGTTC CTAATTTACC ATCTCAGCCA

601 AACTTGGTTT TATCAGACAT AGATAAGTCT CTTATGACCA GCATGGCTGT GACCGCTATG

661 GAAGAATTGC TTAGGCTCAC ACAAACAAAT GAGCCTCTAT GGATCAAAAC CGATGGATGC

721 AGAGAAATTC TCAATCTCGA AAGCTATGAG AATATATTCT CAAGATCAAG TAGCCGTGGA

781 GGAAAGAACC ATAACC TCG AAAGGAAGCA TCTAGATTTT CTGGGGTTGT TTTCACTAAT

841 GCTATGACCC TTGTTGATAT GCTCA GGAC TCGGTCAAAT CTGCAGAGCT TTTTCCCTCA

901 ATTGTTGCAA CATCIAAAAC CCTTGCAGTG ATTTCATCCA GCTTGCGCGG AAACCGTGCA 961 GA GCATTGC ATTTGATGCT TGAAGAGCTT CAAGTGCITT CCCCATTGGT ACCGAGGCGT 1021 GAGTTCTGCG GGCTAAGATA TTGTCAGCAA ATACAACA G GAACTTGGGC AATTGTAAAT 1081 GTCTCCTATG AGTTCCCTCA GTTTATATCT CAT CTCGGT CGTATCGGTA TCCTTCTGGT 1141 TGCTTGATTC AAGACATGTC CAATGGCTAT TCCAAGGTTA T TGGGTCGA ACATGTTGAT 1201 ACGGAGGAGC AAGAACTGGT TCACGAGATG TTTAAAGATA ATGTCCGTCA AGGATTAGCT 1261 TTTGGAGCTG AACG TGGAT GGCTACTCTC CAAAGAATGT GCGAGAGATT CAAGACTCTA 1321 CTAGAACCCG CAACATCTTC CTGCGATCTC AAAGGAGTGA TTCCATCTCT GGAAGGGAAG 1381 AGAAGTATAA TGAGACTATC TCAAAGAATG GTAAGCAACT TTGC TGAG CGT GGCACA 1441 TCTAACAACA ATCGCTCAAC GGTTATTTCC GGGTTGGAGG AGTTTGGAAT CCGTGTGACC 1501 TCGTATAAGA GTCAGCATGA ACCAAA GGA ATGGTTCTAT GTGCAGCCAC TAGTTTCTGG 1561 CTCCCAATTT CTCCACAAAA GGTCTTCAAT TTCCTCAAAG ATGAAAGAAC TCGGCCACAG 1621 TGGGACGT CT TCGAATGG AAATTCTGTT CAAGAAGTTG CTCATATCGC AAACGGCTCA 1681 CACCCTGGAA ACTGCATTTC GGTTCTCTGC GGATTCAA G CATCTTCATC ACAAAACAAC 1741 ATGCTGATTC TACAAGAAAG CTGCGTAGAC TCATCAGGCT CGCTTGTGGT CTACACTCCG 1801 GTGG ATCTAC CAGCACTGAA CATGGCAATG AGCGGTCAAG ACACATCTTA TGTTCCCATT 1861 CTACCCTCGG GT TCGCCGT TTCACCAGAC GGAACCAGGA ATAACCAGCT CGCAGAGCAC 1921 AAAGTTGAAG GAGGAGGAGG TTCATTGATA ACGGT GGTT TTCAGATAAT GGTGAGTAGT 1981 TTGCAGTCAG CGAAAT GAA GTGGAGTCA ATGGAAACAG TTAATAATCT CATCAGCACC 2041 ACTGTCCACC AAATTAAAAC CACCTTGAAC TGTCCTTCAA CTGCTTGA Example 3 7¾HZ) embodiment 6ZHV? Plant gene 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 containing the double enhancer of the ΝΡΤΠ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The 35S promoter containing the double enhancer and the terminator Tnos were selected as promoters and terminators of the ThHDbZIP-3 gene, respectively.

Primer SEQ ID NO: 19 and SEQ ID NO: 20 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ ρΒΙ121, 1.0 μΐ PrimeSTAR, 10 μΜ primer SEQ ID ΝΟ: 19 and SEQ ID NO: 20 each 2.0 μl, and 31 μΐ 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), and extension at 72 ° C for 10 minutes. The resulting PCR product was digested with EcoRI and Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase cassette) to obtain pCAMBIA2300-1.

SEQ ID NO: 19:

GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 20:

ATCCAGATCTAGATCCGGTGCAGATTATTTG

SEQ ID NO: 21 and P SEQ ID NO: 22 amplifies Tnos with pBI121 as a template, using TaKaRa

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 Prime STAR, 10 μΜ of primers SEQ ID NO: 21 and P SEQ ID NO:

22 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), extension at 72 ° C for 10 minutes. The resulting PCR product was digested with Sacl and EcoRI and ligated into the pCAMBIA2300-lCPromega T4 ligase cassette to obtain pCAMBIA2300-2.

SEQ ID NO: 21:

AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 22:

TCAGAATTCCCAGTGAATTCCCGATCTAGTA SEQ ID NO: 23 and SEQ ID NO: 24 Amplify the Arabidopsis thaliana 35S promoter using the pCAMBIA2300 plasmid as a template. PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 μΙ ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ diluted 50-fold pCAMBIA2300 plasmid, 1.0 μΐ PrimeSTAR, 10 μΜ primer SEQ ID NO: 23 and P SEQ ID NO: 24 2.0 11, 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 50 ° C for 30 seconds, elongation at 72 ° C for 30 seconds;), extension at 72 ° C for 10 minutes. The resulting PCR product was ligated by HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2 obtained pCAMBIA2300-3

SEQ ID NO: 23:

ACTAAGCTTATGGTGGAGCACGACACTCT SEQ ID NO: 24:

TGACTGCAGAGAGATAGATTTGTAGAGAGAGAC

SEQ ID NO: 25 and SEQ ID NO: 26 Amplified ThHDbZIP-3 (template was the positive ThHDbZIP-3-pGEM plasmid obtained in Example 2) using Strafugene's PfuUltra II Fusion HS DNA Polymerase. 50 μΐ PCR reaction system: 5 μΐ lOxPfuUltra II reaction Buffer, 0.5μ1 25 mM dNTP, 2.0 μΐ ThHDbZIP-3-pGEM plasmid, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase, 10 μΜ primer SEQ ID NO: 25 and P SEQ ID NO: 26 each of 2.0 μl, and 37.5 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation at 95 °C for 25 seconds, annealing at 58 °C for 25 seconds, extension at 72 °C for 1 minute, extension at 72 °C for 5 minutes. Digestion by Sall, Smal The resulting PCR product was ligated (ligation method as above) to pCAMBIA2300-3 to obtain a plant expression vector 35S-73⁄4HZ1⁄2Z P-3-2300.

SEQ ID NO: 25:

GCGTCGAC ATGGAGTTTCTCGGCGACAG SEQ ID NO: 26:

CCCCCGGGTCAAGCAGTTGAAGGACAGTTC Example 4 35S-ThHDbZIP-3-23 Expression Vector Transformation of Agrobacterium

Agrobacterium LBA4404 (purchased 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 μ _§ /ιη1 streptomycin, and cultured overnight (about 12-16 hours) to OD ₆ at 28 °C with shaking. _{The K)} value is 0.4, and a seed bacterial liquid is formed. 5 ml of the activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium of the same concentration of antibiotic, and cultured at 28 ° C for 2 to 2.5 hours until OD _{6 (K)} = 0.8. The ice bath liquid was shaken for 10 minutes every 3 minutes to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 minutes at 4 ° C, discard the supernatant; add 10 ml of ice-cold 10% (v / v) glycerol resuspended cells, centrifuge at 4000 g for 10 minutes at 4 ° C, collect the precipitate; Ice pre-cooled 10% (v/v) glycerin was washed 3-4 times repeatedly; added 4 ml ice bath pre-cooled 10% (v/v) glycerol to resuspend the bacterial pellet and dispense it at 40 μΐ/tube. Store at -70 ° C for later use.

Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μM of the positive 35S-73⁄4HZ1⁄2Z/P-3-2300 plasmid obtained in Example 3 was added to 40 μM of competent cells, and the mixture was mixed and ice-cooled for about 10 minutes. The mixture of competent cells and plasmid DNA was transferred to an ice-cold electric shock cup with a pipette, and tapped 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. Using a 0.1 cm size electric shock cup, the program of MicroPuMUer (purchased from Bio-Rad) was set to "Agr" and the shock was applied once. Immediately remove the electric shock cup and add 1 ml of LB medium pre-warmed at 28 °C. Quickly and gently spread the cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 225 rpm for 1 hour at 28 °C. 100-200 μL of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 μ _§ /ιη1 rifampicin, 50 μ _§ /ιη1 streptomycin, 50 μ _§ /ιη1 Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Using Agrobacterium-mediated Transformation to Obtain Transgenic Arabidopsis

Plants to be transformed: Arabidopsis seeds (Columbia type, Arabidopsis thaliana Bioresource Center, Ohio State University) Seeded in peat soil, treated at 4 ° C for 3 days, placed at 23 ° C, 16 hours light Sprouting in an 8 hour dark incubator. After 7 - 10 days, transplanted into a plastic pot with a diameter of 7.5 cm containing peat and vermiculite (3:1 by volume), 6 plants per pot, placed at 23 ° C, 16 hours light / 8 hours dark Growing in the incubator. 40 ml of nutrient solution per pot before transplanting, and the soil moisture should be replenished in time after transplanting. The nutrient solution is properly watered during the growth period. Every 3-4 weeks (or longer) as needed. In order to obtain more flower buds on each plant, when the first inflorescence of most plants is formed, the first inflorescence is cut off, and the apical dominance is removed, prompting the simultaneous emergence of multiple secondary inflorescences. Prepare for dip when most of the inflorescences are about 1 - 10 cm high (4-8 days after the first inflorescence is cut).

Culture of Agrobacterium: After removing the bacterial solution of the Agrobacterium-positive transformed clone preserved in Example 4, a single colony of Agrobacterium was picked and inoculated into 10 mL of sterile LB liquid medium (containing 75 mg/L rifampicin). 100 mg/L streptomycin and 100 mg/L kanamycin were shaken overnight at 250 rpm at 28 °C. Then, the obtained bacterial liquid was inoculated into 200 mL of LB liquid medium containing the above antibiotics at a ratio of 1% - 2% (v/v), and the concentration of Agrobacterium was reached at OD _{6 (at a} constant temperature of 28 °C). _K) = 1.8, then centrifuge at 3000 rpm for 15 minutes at 4 ° C, discard the supernatant and use the dip medium (the infusion medium contains 5.0% (w/v) sucrose and 0.05% (500 μΙΤ θ Silwet L-77) Resuspend Agrobacterium and suspend it to 0D _{6 (K)} about 0.80.

Inflorescence dip dyeing: The above Agrobacterium-containing dyeing medium was added to a large-mouth container, and 200-300 mL of the Agrobacterium-containing dyeing medium was added to each container having a diameter of 9 cm for dip dyeing. Invert the plants so that all the aboveground tissues are immersed in the Agrobacterium suspension for 3 - 5 seconds and gently agitate. There should be a liquid film on the plant after infiltration. The immersed plants are placed in a plastic tray, covered with a clean plastic or plastic wrap to moisturize, and then placed in low light or dark overnight, taking care to prevent direct sunlight from the plant. Remove the cover approximately 12 - 24 hours after processing. The plants are cultured normally, and the plants are further grown for 3 - 5 weeks until the pods are browned and dried. Seeds were harvested and the seeds were dried in a centrifuge tube at 4 °C.

Screening of transgenic seeds: Prepare an aqueous solution containing 1/4 MS of large elements, add 0.8% agar powder, heat in a microwave oven until the agar is completely dissolved, and cool to about 50 °C. Add the required amount to a final concentration of 50 rn U ¹ Kanamycin, shake well, pour 25 mL into each dish, and set the bench to cool and solidify. Pour the weighed seeds onto a piece of plain copy paper, tap the copy paper with your fingers, evenly sow the seeds on the agar gel, cover the Petri dish, and cool the chamber at 4 °C for 72 hours, then move to Germinating in a 23 ° C, 16 hour light / 8 hour dark incubator, regular seed germination and seedling growth, and timely transplanting the resistant seedlings into the nutrient soil. After transplanting, the soil moisture is added to the water in time. The nutrient solution is properly watered during the growth period. 0.1 g of leaves of Arabidopsis thaliana grown for 20 days, DNA was extracted, and ThHDbZIP-3 was amplified with SEQ ID NO: 16: and SEQ ID NO: 17 (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 primers SEQ ID NO: 16 and SEQ ID NO: 17 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes 33 cycles (denaturation for 45 seconds at 94 °C, annealing for 45 seconds at 58 °C, extension of 90 seconds at 72 °C), extension at 72 °C for 7 minutes), numbering the plants identified as positive by PCR (T101-T1012), And save. Example 6 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis T1 plants overexpressing ThHDbZIP-3

The sterilized vermiculite was soaked in 1/2 MS medium. T101-T106 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 °C, 10 hours light culture/14 hours dark culture cycle, 1/2MS every 7 days, after 20 days of culture 4-5 seedlings of uniform size were kept in each pot for drought experiments. Transgenic Arabidopsis thaliana, control Arabidopsis thaliana 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 TO transgenic plants) showed that the control plants were wilting, while 26 strains of T101, Τ102, Τ103, Τ104, Τ105 and T106 were each. 22 out of 4) Arabidopsis thaliana survived and continued to grow with obvious drought tolerance (see Figures 3a and 3b, using T106 as an example, T101, T102, T103, TIC 4, T105 results and T106 is similar, not shown here).

Example 7 Determination of ABA changes after drought stress

ABA is recognized as a plant hormone associated with stress, which can act as a signaling molecule to regulate the expression of multiple stress-inducing genes, thereby improving the plant's resistance to stress. We took about 0.2 g of transgenic plants (T101, Τ102, Τ103, Τ104, Τ105, T106) and control plants (CK) under drought stress for 10 days and normal growth conditions, and ELISA with phytohormone abscisic acid (ABA). (ELISA) kit (purchased from Shanghai Ruigu Biotechnology Co., Ltd.) to determine the ABA content (see Figure 4). The results showed that the ABA content of the transgenic plants was higher than that of the control (CK) under drought treatment or control conditions, which proved that the ThHDbZIP-3 gene can positively regulate the endogenous ABA of plants. Example 8 verified the ThHDbZIP-3 protein at the transcriptional level. Expression

Control Arabidopsis thaliana plants, drought-tolerant transgenic Arabidopsis thaliana T1 plants (T101, Τ102, respectively)

Τ103, Τ104, Τ105, T106 (six lines) and the drought-tolerant transgenic Arabidopsis thaliana plants were treated with 0.05 g of leaves per day for 10 days, and total RNA was extracted using the 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 Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (2 μ _β total RNA as a template, reverse transcription primer SEQ ID NO: 18). The relative expression of HDbZIP-3 protein was detected by amplification of ThHDbZIP-3 by SEQ ID NO: 16 and SEQ ID NO: 17.

The PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR 10 μΜ primers SEQ ID NO: 16 and SEQ ID ΝΟ: 17 each 2.0 μl, and 30 μΐ Double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 29 cycles (94 ° C denaturation for 45 seconds, 58 ° C Anneal for 45 seconds, 72 ° C for 2 minutes), and extend at 72 ° C for 10 minutes.

The electrophoresis results of the product are shown in Figure 5: M is DNA Ladder Marker (DL2000, TakaRa), and 1-6 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant (in order: T101, T102, T103, T104, T105, T106).

7-11, is a drought-tolerant transgenic Arabidopsis T1 plant, and 12-16 is a non-transgenic Arabidopsis control. The size of the electrophoresis band of the PCR product shown in the figure is the same as the size of the 73⁄4HZ1⁄2Z/P-3 (about 2.1 Kbp). The results showed that there was no ThHDbZIP-3 transcription in Arabidopsis thaliana, and the transcription of ThHDbZIP-3 was stronger in T1 generation plants of drought-tolerant transgenic Arabidopsis thaliana plants, and the transcription of transgenic Arabidopsis T1 plants was weak.

Claims

claims

1. A leucine zipper protein from Salina chinensis, its sequence is SEQ ID N0: 1.

2. The gene encoding the leucine zipper protein of SEQ ID NO: 1, preferably its sequence is SEQ ID NO: 2.

3. A recombinant expression vector, which contains the gene according to claim 2, and the nucleotide sequence of the gene is operably connected to the expression control sequence of the basic vector used to construct the recombinant expression vector. ; Preferably, the basic vector is pCAMBIA2300.

4. The recombinant expression vector according to claim 3, which is the 35S-7¾HZ½Z/P-3-2300 vector shown in Figure 2.

5. A recombinant cell containing the gene of claim 2 or the recombinant expression vector of claim 2 or 3; preferably, the recombinant cell is a recombinant Agrobacterium cell.

6. A method for improving plant drought tolerance, 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 in question is Arabidopsis thaliana.

7. A method for preparing transgenic plants, comprising: cultivating plants or plant tissues containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions that are effective for producing plants.

8. The method of claim 7, wherein the plant is Arabidopsis thaliana.

9. The use of the gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 4 for improving plant drought tolerance and for plant breeding.

10. The use of claim 9, wherein the plant is Arabidopsis thaliana.