WO2014082285A1

WO2014082285A1 - Cotton zinc finger protein (czf5) and coding gene and use thereof

Info

Publication number: WO2014082285A1
Application number: PCT/CN2012/085628
Authority: WO
Inventors: 崔洪志; 梁远金; 田大翠
Original assignee: 创世纪转基因技术有限公司
Priority date: 2012-11-30
Filing date: 2012-11-30
Publication date: 2014-06-05
Also published as: CN104812900A

Abstract

Provided are a zinc finger protein (Czf5) from cotton and the coding gene thereof, and the use thereof in the incubation of transgenic plants with increased drought resistance.

Description

Cotton zinc finger protein (Czf5) and its coding gene and application

TECHNICAL FIELD The present invention relates to plant proteins and their coding genes and applications, and more particularly to a cotton-derived zinc finger protein (Czf5) and a gene encoding the same, and its use in breeding transgenic plants having improved drought resistance. BACKGROUND OF THE INVENTION Abiotic stresses, such as drought, salting, extreme temperatures, chemical pollution, and oxygen damage, can cause serious damage to the growth and development of plants and cause significant losses to crop yields. 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 areas account for about 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 350-40 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 spring droughts frequently reach 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. 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 promotes 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, DREB1 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; KANG JY. 2002. Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell, 14: 343-357; ABE H. 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as l Transcriptional activators 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. Research on the function and expression regulation of stress-responsive genes will provide an important basis for the study of plant stress-resistance related signaling pathways and signal transmission network systems. SUMMARY OF THE INVENTION The present inventors cloned a zinc finger protein of cotton (designated herein as Czf5) using SSH (suppression subtractive hybridization) and RACE (rapid amplification of cDNA ends), and determined the DNA thereof. sequence. It has also been found that the transgenic plants can significantly improve the drought resistance of transgenic plants by introducing them into plants, and these traits can be stably inherited.

The first aspect of the invention provides a gene encoding a zinc finger protein Czf5 of cotton (this article is named

GhCzf5), 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-Gh Czf5-2300 vector 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 resistance 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 expressing the gene; 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 tissue 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 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 resistance of a plant and for use in plant breeding Use; Preferably, the plant is tobacco.

The seventh aspect of the present invention provides the gene-encoded protein of the first aspect of the present invention, which has an amino acid sequence as shown in SEQ ID NO: 1. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a construction flow of a plant expression vector (rd29A-GhCzf5-2300;) of GhCzf5. Figure 2 is a plasmid map of the plant expression vector Crd29A-GhCzf5-2300 of GhCzf5.

Figure 3 shows the drought resistance growth of control tobacco and transgenic tobacco. CK (left): control tobacco; T1D1 (middle) and T1D8 (right): transgenic tobacco lines.

Figure 4 shows the results of verification of transcriptional levels of T1 transgenic tobacco plants resistant to drought and drought. M is Marker, 1-8 is a drought-tolerant T1 transgenic tobacco plant, and 9-12 is a drought-tolerant T1 transgenic tobacco plant.

BEST MODE FOR CARRYING OUT THE INVENTION The following examples are provided to facilitate a better understanding of the present invention by those skilled in the art. The examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 Cotton SSH library construction under drought stress:

The specific method is:

The method of PCR-select ^TM cDNA Subtraction Kit from Clontech shown by using the subtractive hybridization method suppression construct subtraction libraries. The mRNA extracted from the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA extracted from the leaves of the untreated cotton seedlings was used as a driver. The specific steps are as follows:

(1) Test materials:

African cotton (National Cotton Medium Term Bank, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) was sown on sterilized vermiculite at 25 ° C, photoperiod 16 h / 8 h (light intensity 2000 - 3000 Lx) conditions Under the culture, 1/2MS medium (containing 9.39 mM KN0 ₃ , 0.625 mM KH ₂ P0 ₄ , 10.3 mM NH ₄ N0 ₃ , 0.75 mM MgSO ₄ , 1.5 mM CaCl ₂ , 50 μΜΚΙ, 100 μΜΗ ₃ ΒΟ ₃ , 100 MMnSO ₄ per week) , 30 μΜ ZnS0 ₄ , 1 μΜΝα ₂ Μο0 ₄ , 0.1 μΜ CoCl ₂ , 100 μΜ Na ₂ EDTA, 100 MFeSO 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 two groups, each with 4 pots and 1 pot per pot. The first group was a control group, which was cultured at 25 ° C, photoperiod of 16 h/8 h (light intensity 2000-3000 Lx), and was normally watered. The second group was the drought treatment group, cultured at 25 °C, photoperiod 16h/8h (light intensity 2000-3000 Lx), stopped watering, treated for 10 days, and cut 1/3 of the top of the two seedlings in time after treatment. The leaves, which were rapidly frozen with liquid nitrogen, were stored in a -70 ° C refrigerator.

(3) Total RNA extraction:

Take 0.5 g of cotton leaves from the control and drought-treated groups, respectively, using the plant RNA extraction kit (purchased from Invitrogen) Extracts total RNA from cotton leaves. The absorbance of total RNA at 260 nm and 280 nm was determined by HITACHI's UV spectrophotometer U-2001. The OD _26Q / OD _28Q ratio was 1.8-2.0, indicating a high total RNA purity with 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 purification of polyA+ 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. Clontech's PCR-Select cDNA Subtraction Kits were used to reverse-transcribe driver mRNA and Tester mRNA to obtain double-stranded cDNA, and subtraction hybridization using 2 Tester cDNA and 2 ig Driver cDNA as starting materials. . The Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 h 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 an excess of Driver for the first forward subtractive hybridization. The products of the two first subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the freshly denatured Driver cDNA, and the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.

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

The second PCR product of the 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 For the vector ligation, the specific steps are as follows: The following components were sequentially added using a 200 μΐ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 μ1, 2><Τ4 ligase buffer 5 μl, pGEM-T Easy vector 1 μl, T4 DNA ligase 1 μl, ligated overnight at 4 °C. 10 μΐ of the reaction product was added to 100 μΐ of competent E. coli JM109 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 seconds, ice bath for 2 min, and additional 250 μΐ LB medium (containing 1%). Tryptone (purchased from OXOID), 0.5% Yeast Extract (purchased from OXOID), 1% NaCl (purchased from Sinopharm)), placed in a 37 ° C water bath, shaken at 225 rpm for 30 min, and inoculated with 200 μM bacteria in 50 g/ml Ampicillin was cultured on LB (ibid.)/X-gal/IPTG (X-gal/IPTG purchased from TAKARA) plates at 37 ° C for 18 h. Count the number of clear white and blue colonies > 1 mm in diameter in the culture plate and randomly pick 540 white colonies (number: Gh-D2-001 to Gh-D2-540). All white clones were inoculated into 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 μ _§ /ιη1 ampicillin, 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-select ^TM cDNA Subtraction Kit kit) were used to carry out PCR amplification of the broth, and 452 positive clones were obtained, and then all the positive clones were sent to the British 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 405 effective ESTs (unigene) were obtained.

' : Example 2 Cloning of cotton zinc finger protein gene GhCzf5

After the cloned Gh-D2-196 was detached from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the protein encoded by the sequence belonged to the zinc finger protein. The full-length coding gene corresponding to SEQ ID No: 3 was named GhCzfS. The corresponding protein was named Czf5.

SEQ ID Xo: 3

_ :GAT'J IA CA^CG^: AGTCAACC G AGCATGA TG TCAGTTIGC CT'H G 二ί_ r ^Ai^OA ^ A CA A3A C AACC :: ; ! CT^C GTCA T TGTTTCA: AASSTATSC —

_8_ C :J :G TC !GCCTAC TC H AG G A A TT T ii TT TTCCAT

Cloning of the full-length coding gene of GhCzf5

Based on SEQ ID No: 3 which has been obtained, the following two specific primers were designed as 5, end-specific primers for 3' RACE.

GhCzfo GSP1: SEQ ID NO: I:

GhCzfo GSP2: SEQ ID NO: .5:

The experimental procedure was carried out according to the kit instructions (3, RACE System for Rapid Amplification of cDNA Ends kit was purchased from Invitrogen).

Ffl SEQ ID NO: 4 and 3' primers AUAP (provided with the kit), the first round of PCR amplification was performed using cDNA reverse transcribed cDNA as a template. The specific steps are as follows:

50 μΐ PCR reaction system: 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA inverted cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 4 and AUAP 2.0 1、1, and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 min, 33 cycles (94 °C for 30 s, 58 ° C for 30 s, 72 ° C for 1 min), 72 ° C for 10 min.

The resulting PCR product was diluted 50-fold with double distilled water and 2.0 μL was used as a template to replace the page with SEQ ID NO: 5 and 3' (Rule 26) The first primer AUAP performs the first round of PCR amplification, and the specific steps are as follows:

50 μΐ PCR reverse;, V: system: 5 μΐ ΙΟχΕχ 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: 5 and AUAP 2.0 1、1, and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (94'C denaturation for 30 s, 58 °C for 30 s, 72 °C for 1 min), 72 °C for 10 min. A strip of about 500 bp in the second PCR product was recovered (Gel Extraction Kit purchased β OMEGA), and it was ligated to pGEM-T Easy Vector, and then transformed into the large intestine rod JM109 (the specific method is the same as above), and randomly picked. Ten white colonies were inoculated in LB liquid medium containing 50 g/mL ampicillin, cultured at 37 ° C overnight, and th oil was added to a final concentration of 20%, and stored at -80 ° C until use. Using SEQ ID NO: 5 and the 3' primer AUAP for sputum PCR amplification, 9 positive clones were obtained, and 4 positive gram clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing. The 3' end of the cDNA of the gene.

Based on the GhCzf5 gene fragment obtained, the following three specific primers were designed as the 3'-end specific primer of 5' RACE.

GhCzfS GSP3: SEQ ID NO: 6:

GAftAAT TCC GCA CTCAC AA

GhCzfS GSP I : SEQ ID NO : 7:

TT GCAGC AGAG GTT C TG

GhCzfS GSP5: SEQ ID NO: 8:

GCA GT ACA TT AATAGT TC Experimental procedure was performed according to the reagent 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: 7 and 5' universal primer AAP (provided with the kit), and cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification. The specific steps are as follows:

50 μΐ PCR reaction system: 5 μΐ 10><Ex Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΙ Εχ Taq (purchased from TAKARA), 10 μΜ primer SEQ ID NO: 7 and AAP each with 2.0 μl and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (94'C variability 30 s, 55 'C annealing 30 s, 72 °C extension lmin), 72 "C extension for 10 min.

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: 8 and the 3' primer AUAP, and the specific steps were as follows:

50 μΐ PCR reaction: 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first

6

Replacement page (Article 26) Round PCR product, 1.0 μΐ Ex Taq, 10 μΜ primers SEQ ID NO: 8 and AUAP each 2.0 μl, and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min. Recovering the fragment of the second PCR product is approximately

H H

500 bp strip H (Gel Extraction Kit from OMEGA), and connected to pGEM-T Easy Vector,

: '

Transformed to JM109 (the same method as above), randomly picked 10 white colonies inoculated with 50 μ /ηι1 ampicillin

H

i” ■

In the LB liquid medium-V', cultured at 37 ° C overnight, glycerol was added to a final concentration of 20%, and stored at -80 ° C for use. Using SEQ ID NO _: 8 and the 3' primer AUAP to carry out bacterial PCR amplification (reaction system and reaction conditions as above),

H H

Nine positive clones were selected, and four of them were selected. (:! The clone was sent to Yingjie Jieji (Shanghai) Co., Ltd. for sequencing and sequencing, and the 5' end of the cDNA was obtained.

F

The resulting 5' RACE product was cloned and sequenced, and ligated with the 3' RACE product sequencing result and the SEQ ID No: 3 sequence. The full-length cDNA sequence of GhCzf5 was obtained SEQ ID No: 9:

SEQ ID NO: 9:

T AA'STCT^ AG C ATC AG AGGASCG _ TCTTGGTAAA T CATAT^TA T3 TTAAA53 CAIiJiTT GA

- A ATTATC G -::: C^ CCT CGTl'C ^ C :: C:.:'TCCT::C

- - ATCAT. T S C ACC CGG A CATTGA AATC CTG

_ 3⁄4 _ ^"" ¹ c■? GA AGT ACA TA AGA TT "C GA'^CAS : A^ G^ T AAC GA^CA. T ACTGTT A ; GITTGC TA GAGATTAAC GCT GTC TG CGGT^ACG

AA CATTGGA ATGTCACA G : C T TTT C ::'― G-:C A.:'TT7T QGT AG GT CSGA A TT?

A TACAG ^: TA T ^: QT AA GT TTT AAA AAAC GAT'S CGC^C 一·"_; -ACCCI GA'S ATGTGGT:3⁄4 G?TT ? TT^

'': ':― CTGAAGTTGT ATG. ?CATC CACTTA GTT

■■'― - AT '■_";_- TTTTCGA-TA GTGSAAT AA AATCTGCT A

―:;- AG TGTTG T GT^ AAT T ATAATGT A

n

::4. T A.TTTTTT AT -iC-TATTI:: TT CAATTTA TTTAAAAA AAAAAAAAAA

:■ _ A pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows: GhCzfo: 3EQ ID NO: 10:

AT^ GT.:CC TCAASCCT C A

GhCzfS: SEQ ID XO: U:

One - - ' - one

7

Replacement page (Article 26) The GhCzf5 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11. The PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template. 50 l PCR reaction system: 10 ^ 5xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 μ1, and 30 μΐ double steamed water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.

The PCR amplification product plus A tail: 2.5 times the volume of absolute ethanol was added to the PCR product, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, air-dried, and then dissolved in 21 μl of double distilled water. Then, 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μΐ 5 mM dATP, 2.5 μΐ ΙΟχΕχ Taq were added thereto. Reaction conditions: 70 'C reaction for 30 minutes. The obtained DNA fragment of about 500 bp was recovered (Omega recovery kit), and ligated into pGEM T-easy vector, and then transformed into JM109 (method as above), and 10 white colonies were randomly picked and inoculated to contain 50 g/mL. Ampicillin in LB liquid medium, cultured at 37 ° C overnight, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. The bacterial liquid PCR amplification was carried out with SEQ ID NO: 10 and SEQ ID NO: 11 (reaction reaction and reaction conditions are the same as above), and Ί positive clones were obtained, and 4 positive clones were selected and sent to Yingjiejie (Shanghai) Trade. Sequencing, the sequence is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1.

Amino acid sequence of Czf5 protein: SEQ ID NO: 1

G1SSLPAPS EGVLCVLLVK

TALSISMVK

"HLSSSSSSS SSSSSS3SSS

SSSPSS LIZ IPAVSFDISI

GNA SYIKEF RS MPST YD

AVCS SQFEH DCSVCLTRFH:

PESEIKRLS GHLFHKVCLS

Ir" ϊ^. i* >― >

Nucleotide sequence of the GhCzf5 encoding gene: SEQ ID NO: 2

8 Replacement page (Article 26) 丄

TATG GIACT CT GGTAAAC . ^ A^ TTT CCAIAT TAT G - "IAAAGG ATAATT SAT CAAT C TCA CATTAT GT 丄::丄AT:: AT TCT CC TCCTC: JTC T CC TC rCCT CT CCTCGTCA C A-OATCAT A i-Si ZZKICh ZQC CAT CCG ATT AT7GAA ATCCCI-S»: 5S AT ATTCGA: A CAGCATC II 4_ G: iTAATG: TG iTA'JTCACAC TAAAGAGTT CG G AGGA TGCCAT GAC C GGTACGAT

30 _ ί.3ΰ ΑΙ33⁄4 G GS AGT AA CGiAGCAT GA TT CAG TT CCTGAC TAGGTTCGAG 3€1 CA AAT A^ A^AITAA : TT T:CG: GGTCAT^ T CACAAG T A7 CTTG:SAA 2^ AA TGGTTGA AC ATTG JAA TGTCACATGC CCTCTTTGCA GGAACG TCT : 5CTSCCC5AA

46_ : CTA TTTG ^ GA

Example 3 Construction of GhCzf5 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 containing the double enhancer of the ΝΡΤΠ gene was replaced with the Pnos promoter to reduce the expression of NPTII protein in plants. . The inducible M29A promoter and terminator Tnos were selected as promoters and terminators of the GhCzf5 gene, respectively.

Pnos were amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using primers SEQ ID NO: 12 and SEQ ID NO: 13, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μl ΡΟ Reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ ρΒΙ12Κ 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 12 and SEQ ID NO: 13 each 2.0 μl, and 31 μl double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s), and extension of 72'C for 10 min. The resulting PCR product was cleaved by EcoRI and Bglll to pCAMBIA2300 (promega, T4 ligase cassette) to obtain pCAMBIA2300-1.

SEQ ID XO: 12 :

GCACG TTCATACAAATGGACGAACGGAT SEQ ID NO: 13:

ATCCAGATCTAGATCCGGTGCAGATTATTTG

Tnos was amplified using primers SEQ ID NO: 14 and SEQ ID NO: 15 with pBI121 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, 1.0 μΐ pBI12 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 14 and SEQ ID 1^0: 15 each 2. (^1 And 31 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C 10 Min. The resulting PCR product was ligated by Sacl and EcoRl (promega T4 ligase cassette) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.

Replacement page (Article 26) SEQ ID -, Ό: 11:

AAG (H AATTTCCCCGATCGI TCAAA

SEQ ID NO: 15:

TCA^ f CAGTGAATTCCCGATCTAGTA

Amplification of Arabidopsis thaliana rd29A with primers SEQ ID NO: 16 and H SEQ ID NO: 17 was initiated with Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) DNA (see Zeng J., et Al. 2002, Preparation of total DNA from "recalcitrant plant taxa", Acta Bot. Sin., 44(6): Method 694-697 for extracting Arabidopsis DNA). 3⁄4 uses TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reverse v: system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ Arabidopsis DNA, 1.0 μM PrimeSTAR > 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 2.0 μ1, and 31 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), and extension at 72 V for 10 min. The resulting PCR product was ligated by HindIII and Pstl (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.

SEQ ID NO: 16:

ACTAAGCTTCCTTCTTGACATCATTC TTTTA SEQ ID NO: 17:

Ding GAr7 ( UCCAAAGATTTTTTTCTTTCCAATAG

The full-length sequence of the GhCzf5-encoding gene was amplified using primers SEQ ID NO: 18 and SEQ ID NO: 19 (model: 3⁄4 GhCzf5 full-length coding gene obtained in Example 2), and PrimeSTAR HS DNA polymerization using TaKaRa

^嗨. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer , 3 μΐ 2.5 mM dNTP, 1.0 μM GhCzf5-pGEM 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 18 and SEQ ID NO: 19

^ 2 0 μΙ, and 3 1 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), 72 "C extension" for 0 min. Passing Pstl, Sacl enzyme The resulting PCR product was ligated (ligation method) to pCAMBIA2300-3 to obtain plant expression ry29A-GhCzf5-2300.

SEQ ID NO: 18:

Ding GAr/3⁄4t^i(i ATGGGT C T AAGTCT C CA

SEQ ID NO: 19:

kAGGAGCTC T A CAAAAG TAGSCCGGAT C STC

Example 4 Transformation of Agrobacterium by rd29A-GhCzf5-2300 Expression Vector

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Replacement page (Article 26) 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 Line 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 h) to OD ₆ at 28 °C with shaking. . A value of 0.4 forms a seed broth. 5 ml of activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 μ _§ /ιη1 rifampicin and 50 μ _§ /ιη1 streptomycin, and cultured at 28 °C with shaking 2 2.5 h to OD _6QQ = 0.8. The ice bath liquid was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state uniformly. Centrifuge at 4000 g for 10 min at 4 °C, discard the supernatant; add a certain amount of ice-cold 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 °C, collect the precipitate; use and pre-cool 10 % glycerol was washed 3-4 times repeatedly; then the bacterial pellet was resuspended by adding 10% glycerol pre-cooled with an appropriate amount of ice bath, dispensed at 40 μΐ/tube, and stored at -70 °C until use.

Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μΐ of the plasmid was added to 40 μΐ of competent cells, and the mixture was mixed and ice bathed for about 10 minutes. Transfer the mixture of competent cells and DNA to a ice-cold electric shock cup (purchased from bio-md) with a pipette and tap to bring the suspension to the bottom of the electric shock cup, taking care not to have air bubbles. Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup to the base electrode of the electric shock chamber. When using a 0.1cm size electric shock cup, the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is applied once. Immediately remove the electric shock cup and add the pre-warmed LB medium 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 28 ° C, 225 rpm for 1 h. 100-200 μL of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 μ _§ /ιη1 rifampicin, 50 g/ml streptomycin, 50 g/ml Kana) Phytomycin), cultured at 28 °C. Example 5 Obtaining Transgenic Tobacco by Agrobacterium-mediated Transformation

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 s, then wash twice with sterile double distilled water. Soak it in 0.1% liters of mercury for 8 min, then wash it twice with sterile double-distilled water to complete surface sterilization. Surface-sterilized tobacco seeds were placed in MS solid medium (containing 18.78 mM KN0 ₃ 1.25 mM KH ₂ P0 ₄ , 20.6 mM H ₄ N0 ₃ 1.5 mM MgS0 ₄ , 3.0 mM CaCl ₂ , 50 μΜ ΚΙ, 100 μΜ Η ₃ ΒΟ ₃ , 100 M MnSO ₄ , 30 M ZnSO ₄ , 1 μΜ Να ₂ Μο0 ₄ 0.1 μΜ CoCl ₂ 100 μΜ Na ₂ EDTA 100 μΜ FeS0 ₄ , 7.4 g/1 Qiongyue, 30 g/l sucrose) Germinating under the conditions of bacteria, preparation of sterile seedlings. The leaves of sterile seedlings were cut into 5 mmx5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing expression vector rd29A-GhCzf5-2300 in logarithmic growth phase for 10 min, and the bacterial culture was sucked up and co-cultured under dark conditions. 2 days (MS solid medium). Transfer the leaves to a differentiation solid medium (MS+1 mg/1 cytokinin (BA) + 0.1 mg/1 naphthaleneacetic acid (NAA) + 50 mg/1 kanamycin + 500 mg/1 cephalosporin) , using 2000 Lx of light for 16 hours per day for about 45 days. After the buds grow up, they are cut and transferred to rooting medium. (MS+50 mg/l kanamycin+500 mg/l cephalosporin) was cultured for about 30 days. After the root system was developed, the seedlings were transferred to MS medium supplemented with 500 mg/1 cephalosporin. The number is saved.

The leaves of the obtained transgenic tobacco plants were cut out, DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and PCR amplification was carried out using SEQ ID NO: 10 and SEQ ID NO: 11 (50 μΙ ΡΟ 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: 10 and SEQ ID NO: 11 each 2.0 μl, and 35 μΐ double distilled water. : 94 °C pre-denaturation 5 min, 33 cycles (94 °C denaturation 30 s, 58 °C annealing 30 s, 72 °C extension 2 min), 72 °C extension 10 min), PCR identified as positive plant number It is T0D1-T0D20 and saved. Example 6 Overexpression of GhCzf5 Transgenic Tobacco T1 Generation Drought Resistance Simulation Experiment

The sterilized vermiculite was soaked in 1/2 MS medium. T0D1-T0D20 transgenic tobacco and control tobacco seeds were separately sown on vermiculite, 15 seeds per pot, 25 ° C, 10 hours light culture / 14 hours dark culture cycle. 1/2MS was poured every 5 days. After 25 days of culture, 4-5 seedlings of the same size were kept in each pot for drought test. Transgenic tobacco and control tobacco were dried for 14 days (no watering), 25 °C, 10 Hour light culture / 14 hour 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 T1D1, T1D8, T1D10, T1D13 and T1D17 had 18 of 23 tobaccos. It can grow normally and exhibits obvious drought tolerance (see Figure 3, taking T1D1, T1D8 as an example. The results of T1D10, T1D13, T1D17 are similar to T1D1, T1D8, not shown here). Example 7 Verification of Czf5 protein expression at the transcriptional level In Example 6, 18 of the 1\ generation transgenic plants capable of growing normally under drought conditions were randomly selected, and 5 of the examples 6 were not able to grow normally under drought conditions. Four of the 1\ transgenic plants were randomly selected, and 0.05 g of leaves with drought for 14 days were cut out. Total RNA was extracted with plant RNA extraction kit (invitrogen). The absorbance values of total RNA at 260 nm and 280 nm were determined by ultraviolet spectrophotometry, and the respective RNA concentrations were calculated. Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (1 total RNA as a template, reverse transcription primer SEQ ID NO: 11). The relative expression of Czf5 protein was detected by amplifying GhCzf5 by SEQ ID NO: 10 and SEQ ID NO: 11. 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 5xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 μl, and 30 μΐ double Steamed water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 29 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min. The product electrophoresis results are shown in Figure 4: M is the DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Biotechnology Co., Ltd.), 1-8 is a normal growing plant, and 9-12 is a plant that cannot grow normally. The strip size shown in the figure is the same as the size of GhCzf5. The results showed that the transcription of GhCzf5 in normal growing plants was stronger, and there was no GhCzf5 transcription or transcription in normal growth plants. Those skilled in the art will appreciate that the present invention is also applicable to variations and modifications other than the specific description, and the present invention includes all such variations and modifications. The inventors claim the right to modify and change within the scope and spirit of the following claims.

Claims

Claim

1. A gene encoding a zinc finger protein of cotton, designated GhCzf5, having the nucleotide sequence shown in SEQ ID NO: 2.

2. A recombinant expression vector comprising the gene of claim 1 and the nucleotide sequence of the gene operably linked to an expression control sequence of the expression vector.

3. The vector of claim 2 which is the rd29A-GhCzf5-2300 vector 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 resistance 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 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 resistance 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.