CN102766610B - Plant drought-resistant relevant protein PvSnRK 2.3 and encoding gene and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular, the invention relates to a plant drought resistance-related protein PvSnRK2.3 and its coding gene and application. The drought resistance-related protein PvSnRK2.3 provided by the present invention is derived from switchgrass, and its amino acid sequence is shown in SEQ ID NO.1. The drought resistance-related protein and its coding gene of the invention have very important theoretical and practical significance for improving and enhancing the stress resistance of Arabidopsis thaliana, increasing yield, accelerating the process of stress-resistant molecular breeding, and effectively saving water resources.

Description

A plant drought resistance related protein PvSnRK2.3 and its coding gene and application

technical field

The invention relates to the field of genetic engineering, in particular, the invention relates to a plant drought resistance-related protein PvSnRK2.3 and its coding gene and application.

Background technique

As one of the important food crops in our country, wheat occupies a very important position in the national economy. However, adversity stress conditions such as drought and salinity reduce the output of my country's wheat by about 80 billion kilograms every year, seriously affecting the yield and quality of wheat, and restricting my country's wheat food security. With the development of modern molecular biology, genetic engineering technology is used to study the relationship between plants and abiotic stress at the molecular level, reveal the molecular mechanism of plant stress signal transduction and gene expression regulation, and provide new ways for cultivating new stress-resistant crops. quality provides a theoretical basis.

In recent years, the identification and elucidation of the mechanism of gene expression regulation under various conditions through the structural and functional analysis of protein kinases has received extensive attention. The sucrose non-fermentation-related protein kinase family (SnRKs) plays an important role in many physiological processes in plants, such as hormone signal transduction, abiotic stress and plant growth and development. SnRK protein kinases belong to the super family of serine/threonine protein kinases, which are divided into three subfamilies due to the similarity of gene sequence and the difference of gene structure: SnRK1, SnRK2 and SnRK3. The three subfamilies of SnRK protein kinases all have similar structural characteristics, and the N-terminal has a kinase domain that can interact with other proteins, and the structure is highly variable among the three families, but it is similar to other protein kinase families. In contrast, this domain domain contains a conserved threonine.

The SnRK2 family genes showed certain differences in function. Among the SnRK family members in Arabidopsis, 9 genes were induced by hyperosmotic stress (mannitol or NaCl), and 5 genes were induced by ABA, but none of them were affected by cold stress. induced. SnRK genes in rice, through protein phosphorylation analysis, show that all members can be activated by hyperosmotic stress, but only three genes, OsSAPK8, OsSAPK9 and OsSAPK10, are induced by ABA. OsSAPK4 is a gene of the rice SnRK2 family. The induced expression of this gene It can improve the germination rate of rice seeds under salt stress and improve the drought resistance of mature plants. The functions of the SnRK2 family genes in wheat are also somewhat different. Overexpression of the TaSnRK2.4 gene in Arabidopsis can significantly enhance the stress resistance of the plant; functional analysis of the TaSnRK2.7 gene shows that it can be used in glucose metabolism, reducing osmotic potential, enhancing The activity of photosystem II plays an important role in the promotion of plant rooting and other physiological and biochemical processes; Arabidopsis overexpressing the TaSnRK2.8 gene has a certain tolerance to drought, low temperature, high salt and other stresses.

In summary, SnRK protein kinases play a vital role in regulating plant stress responses and improving plant stress resistance, and stress-resistant breeding and agricultural production will have a huge boost and economic benefits. Therefore, using the stress-resistance-related SnRK protein kinase gene to improve and improve the stress resistance of crops has very important application prospects.

Contents of the invention

The purpose of the present invention is to provide a plant drought resistance related protein PvSnRK2.3.

Another object of the present invention is to provide a gene encoding the above-mentioned plant drought resistance-related protein PvSnRK2.3.

Another object of the present invention is to provide a recombinant vector comprising the above gene.

Another object of the present invention is to provide recombinant cells comprising the above-mentioned genes.

Another object of the present invention is to provide the application of the above-mentioned plant drought resistance-related protein PvSnRK2.3.

The drought resistance-related protein PvSnRK2.3 provided by the present invention is derived from switchgrass, and its amino acid sequence is shown in SEQID NO.1.

The protein kinase of the present invention consists of 330 amino acid residues and is a SnRK protein kinase. The 10th-30th amino acid residue from the amino terminal of SEQ ID NO.1 is an ATP binding domain, and the 120th-130th amino acid residue from SEQ ID NO.1 is a serine/threonine binding domain.

SEQ ID NO.1

1 MEERYEALKE LGAGNFGVAR

21 LVRDKRTKEL VAVKYIERGK

41 KIDENVQREI INHQSLRHPN

61 IVRFKEVCLT PTHLAIVMEY

81 AAGGELFEKI CSAGRFSEDE

101 SRYFFQQLIS GVSYCHSMEI

121 CHRDLKLENT LLDGSPTPRV

141 KICDFGYSKS ALLHSKPKST

161 VGTPAYIAPE VLSRKEYDGK

181 VADVWSCGVT LYVMLVGSYP

201 FEDPEDPRNF RKTISRILGV

221 QYSIPDYVRV SSDCRRLLSQ

241 IFVSDPSKRI TIPEIKKHPW

261 FLKNLPREIS EREKANYKYT

281 EPAEPAQAVD EIMRIVQEAK

301 TPGDMSKAVD PALLAEMAAL

321 ESDGEEADAD DAY*

In order to make the protein PvSnRK2.3 easy to purify, the amino-terminal or carboxy-terminal of the protein composed of the amino acid sequence shown in SEQ ID NO.1 can be connected with the tags shown in Table 1.

Table 1 Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

According to the sequence of SEQ ID NO.1 disclosed in the present invention, the transcription factor PvSnRK2.3 of the present invention can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression.

The PvSnRK2.3 coding gene according to the present invention has a nucleotide sequence as shown in SEQ ID NO.2.

SEQ ID NO.2

1 ATGGAGGAGA GGTACGAGGC GCTGAAGGAG CTGGGCGCCG GTAACTTCGGGGTAGCGAGG

61 CTGGTCAGGG ACAAGCGGAC CAAGGAGCTC GTCGCCGTCA AGTACATCGAGAGGGGCAAG

121 AAGATTGATG AGAATGTGCA GAGGGAGATC ATCAATCACC AGTCGCTCCGGCACCCTAAC

181 ATCGTACGGT TCAAGGAGGT TTGTTTAACG CCCACACATC TTGCCATTGTAATGGAATAT

241 GCTGCTGGTG GAGAGCTCTT TGAGAAAATC TGCAGCGCGG GGAGATTCAGTGAAGATGAG

301 TCCAGGTATT TCTTTCAGCA GCTAATATCA GGAGTCAGCT ACTGCCATTCCATGGAAATT

361 TGTCACCGTG ATCTTAAACT TGAGAACACT CTCCTTGATG GGAGTCCAACACCTCGCGTG

421 AAAATTTGCG ACTTCGGTTA CTCAAAGTCT GCCTTGCTGC ATTCCAAACCAAAATCGACA

481 GTTGGCACTC CAGCTTACAT AGCGCCTGAG GTTCTTTCCA GAAAAGAGTATGATGGCAAG

541 GTAGCAGATG TTTGGTCCTG CGGCGTGACA CTGTATGTAA TGCTCGTGGGATCATATCCA

601 TTTGAAGATC CCGAGGATCC AAGGAACTTC CGCAAGACGA TCAGCAGAATTCTTGGCGTG

661 CAATACTCCA TCCCGGATTA TGTGAGAGTG TCTTCAGACT GCAGACGCCTTCTGTCTCAA

721 ATTTTCGTCT CCGATCCTTC AAAGAGGATC ACGATCCCGG AGATAAAGAAGCACCCGTGG

781 TTCCTGAAGA ACCTGCCGCG GGAGATCTCG GAGAGGGAGA AGGCCAACTACAAGTACACG

841 GAGCCCGCCG AGCCGGCGCA GGCCGTGGAC GAGATCATGC GGATCGTCCAGGAGGCCAAG

901 ACCCCCGGCG ACATGTCCAA GGCCGTGGAC CCGGCGCTGC TCGCCGAGATGGCCGCGCTG

961 GAGAGCGACG GGGAAGAAGC CGACGCCGAT GACGCCTACT GA

The expression cassette, recombinant expression vector, transgenic cell line and recombinant bacteria containing PvSnRk2.3 gene all belong to the protection scope of the present invention.

The existing plant expression vector can be used to construct the recombinant expression vector containing PvSnRK2.3 gene.

The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene 3' end transcription The untranslated regions of both have similar functions.

When using PvSnRK2.3 to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription start nucleotide, such as cauliflower mosaic virus (CaMV) 35S promoter, maize Ubiquitin promoters (Ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. The enhancer region can be the initiation codon of ATG or the initiation codon of the adjacent region, etc., but it must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) Genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

Another object of the present invention is to provide a method for cultivating stress-tolerant plants.

The method for cultivating stress-tolerant plants provided by the present invention is to introduce any one of the above-mentioned recombinant expression vectors containing PvSnRk2.3 gene into plant cells to obtain stress-tolerant plants.

Using any vector that can guide the expression of foreign genes in plants, the SnRK protein kinase PvSnRK2.3 gene provided by the present invention is introduced into plant cells, and a transgene with enhanced tolerance to abiotic stresses such as drought and salt can be obtained Cell lines and transgenic plants. The expression vector carrying the coding gene can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium-mediated, and transform the transformed plant Tissues are grown into plants. The transformed plant host can be either monocot or dicotyledon, such as: Arabidopsis, wheat, switchgrass, Arabidopsis, rice, corn, cucumber, tomato, poplar, lawn grass, alfalfa, etc. .

The stress tolerance of the plant can specifically be stress tolerance to abiotic stress, such as stress tolerance to or salt stress.

The present invention uses switchgrass (Panicum virgatum L.) with strong drought resistance and salt tolerance as the experimental material, obtains the stress-resistance-related PvSnRK2. drought resistance. The drought resistance-related protein and its coding gene of the invention have very important theoretical and practical significance for improving and enhancing the stress resistance of Arabidopsis thaliana, increasing yield, accelerating the process of stress-resistant molecular breeding, and effectively saving water resources.

Description of drawings

Figure 1 shows the screening of T1 generation PvSnRK2.3 transgenic Arabidopsis lines.

Figure 2 is the PCR identification of transgenic Arabidopsis T1 line, M: Trans2K Plus DNAmarker; 1: negative control; 2: water control. 3-24 are transgenic Arabidopsis lines.

Fig. 3 Germination of transgenic Arabidopsis under different stress treatments.

Fig. 4 Identification of drought tolerance of transgenic Arabidopsis, WT is wild type Arabidopsis, L1, L8, L9 are three PvSnRK2.3 transgenic Arabidopsis lines.

Detailed ways

For the molecular biology experiment methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kits and product instructions conduct.

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

Example 1: cDNA Cloning of PvSnRK2.3 Gene Related to Drought Resistance in Switchgrass.

The 30-day-old switchgrass seedlings were subjected to drought treatment for 5 hours, and the total RNA of switchgrass was extracted with Trizol. Apply 5'RACE kit (5'RACE System for Rapid Amplification of cDNA Ends Kit) (GIBCOBRL, CAT.NO.18374-058) and 3'RACE kit (3'RACE System for Rapid Amplification of cDNA Ends Kit) (GIBCOBRL , CAT.NO.18373-019) obtained the full-length sequence of PvSnRK2.3 gene 1002bp.

The total RNA of switchgrass seedlings was extracted with Trizol, and cDNA was obtained by reverse transcription with superscript II (invitrogen) reverse transcriptase. Primers P1 and P2 were designed according to the sequence of the coding region of PvSnRK2.3 gene. Using the cDNA obtained by reverse transcription as a template, PCR amplification was performed with primers P1 and P2. The sequences of primers P1 and P2 are as follows:

P1: 5'-ATCGTCCAGGAGGCCAAGA-3',

P2: 5'-GCTTTCTTCCCCGTCGCTCT-3'.

The PCR product was detected by 0.8% agarose gel electrophoresis, and a band with a molecular weight of about 1 kb was obtained, which was consistent with the expected result. This fragment was recovered with an agarose gel recovery kit (TIANGEN). The recovered fragment was ligated with pGEM-T Easy (Promega), referring to the method of Cohen et al. (Proc Natl Acad Sci, 69:2110), and the ligated product was transformed into Escherichia coli DH5α competent cells. Positive clones were screened with penicillin resistance markers to obtain recombinant plasmids containing recovered fragments. The T7 and SP6 promoter sequences on the recombinant plasmid vector were used as primers to carry out nucleotide sequence determination, and the sequencing results showed that the open reading frame (ORF) of the amplified PvSnRK2.3 gene was the original sequence of SEQ ID No.2. The 1st to 1002nd deoxyribonucleotides at the 5' end encode a protein whose amino acid sequence is SEQ ID No.1. The recombinant vector containing the PvSnRK2.3 gene shown in the sequence SEQ ID No.2 is named pTE-PvSnRK2.3.

The sequence of PvSnRK2.3 gene was compared on Genabnk. The gene has high homology with SnRK protein kinase in Arabidopsis, but no homologous protein gene was found in switchgrass, which proved that PvSnRK2.3 gene is a new gene.

Example 2: Enhancement of plant drought tolerance with PvSnRK2.3 gene

1. Construction of recombinant expression vector

1) Construction of 35S-PvSnRK2.3 recombinant expression vector

Using the cDNA obtained by reverse transcription of switchgrass total RNA as a template, PCR amplification was performed with specific primers containing SmaI and SpeI linker sequences; then SmaI and SpeI double-enzyme-digested PCR products were recovered, and the enzyme-digested products were inserted forward into the vector pBI121 Between the SmaI and SpeI restriction sites behind the CaMV35S promoter, the recombinant vector p35S-PvSnRK2.3 was obtained.

The primer sequences are as follows:

PvSnRK2.3[SmaI]5'-TC CCCCGG GATGGAGGAGAGGTAC-3'

PvSnRK2.3[SpeI]5'-GG ACTAGT TCAGTAGGCGTCATCGGCG-3'

2. Acquisition and functional identification of transgenic Arabidopsis

1) Obtaining transgenic Arabidopsis

The recombinant expression vector p35S-PvSnRK2.3 constructed above was transformed into Agrobacterium tumefaciens EHA105 by freezing and thawing method, and then transformed into Arabidopsis thaliana with p35S-PvSnRK2.3 Agrobacterium tumefaciens EHA105, with 100 mg/L Kana The positive transgenic plants were obtained by screening on the MS medium of prime. The positive transgenic plants obtained by screening were further identified and screened by PCR, and a pair of primers used in PCR were P3 and P4.

P3 (upstream primer): 5'-ATGGAGGAGAGGTAC-3',

P4 (downstream primer): 5'-TCAGTAGGCGTCATCGGCG-3'.

The 35S::PvSnRK2.3 transgenic Arabidopsis was identified by PCR, and the positive transgenic plants were amplified by PCR to obtain a band of about 1Kb. As a result, 35 35S::PvSnRK2.3 transgenic Arabidopsis plants were obtained (Figure 1, Figure 2) .

At the same time, the pBI121 empty vector was introduced into Arabidopsis thaliana by the same method as above. As a control, 10 strains of empty vector Arabidopsis thaliana were obtained (the transgenic Arabidopsis obtained by screening is represented by T ₃ generation).

2) Statistical analysis of germination rate of transgenic Arabidopsis under ABA and PEG stress

As shown in Figure 3, both transgenic Arabidopsis and wild-type Arabidopsis could grow normally on MS medium. On MS medium, the germination rate of all transgenic Arabidopsis was higher than that of wild-type Arabidopsis. On the 10% PEG medium, the germination rate of the transgenic Arabidopsis thaliana of the two lines L9 and L19 was higher than that of the wild type Arabidopsis, and on the 200mmol L ^-1 medium, the germination rate of the transgenic Arabidopsis of the L8 line The germination rate of the four lines L8, L9, L18, and L19 was higher than that of wild-type Arabidopsis on 50 μM ABA MS medium, and the germination rate of L7 was lower than that of wild-type Arabidopsis Mustard (Figure 3).

3) Identification of drought tolerance of transgenic Arabidopsis seedlings

In order to further test the tolerance of the transgenic Arabidopsis plants to drought stress, the phenotypes of the transgenic Arabidopsis plants on the 0th day, the 10th day, the 25th day and the 5th day after rehydration were photographed and observed. After 25 days of drought treatment, the transgenic line L8 The color of the rosette leaves of these two lines, L9 and L9, became darker, and a few wild-type and transgenic plants died. Then rehydration began on the 26th day. Observation and photography on the 5th day after rehydration showed that the survival rates of wild-type Arabidopsis, L1, L8, and L9 were 5.3%, 77%, 69%, and 30%, respectively. The survival rate of L9 among the three transgenic lines was lower, but higher than that of wild-type Arabidopsis (Fig. 4).

Claims (7)

1. A plant drought resistance-related protein PvSnRK2.3, characterized in that its amino acid sequence is as shown in SEQ ID NO.1. 2. A plant drought-resistance-related gene PvSnRK2.3, characterized in that it encodes the plant drought-resistance-related protein PvSnRK2.3 according to claim 1. 3. The plant drought resistance related gene PvSnRK2.3 as claimed in claim 2, characterized in that its base sequence is as shown in SEQ ID NO.2. 4. A recombinant vector comprising the plant drought resistance related gene PvSnRK2.3 according to claim 2. 5. The recombinant vector p35S-PvSnRK2.3 comprising the plant drought-resistance-related gene PvSnRK2.3 described in claim 2, the plant drought-resistance-related gene PvSnRK2.3 is double-digested with SmaI and SpeI, and the digestion product is inserted into the vector forward Between the SmaI and SpeI restriction sites behind the CaMV35S promoter of pBI121, the recombinant vector p35S-PvSnRK2.3 was obtained. 6. The application of the plant drought resistance-related protein PvSnRK2.3 in claim 1 for enhancing the drought tolerance of plants, wherein the plants are switchgrass or Arabidopsis. 7. The application of the plant drought resistance-related gene PvSnRK2.3 as claimed in claim 2 for enhancing the drought tolerance of plants, wherein the plants are switchgrass or Arabidopsis.

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