WO2008022486A1

WO2008022486A1 - Plant growth and stress tolerance related isozyme, encoding gene and use thereof

Info

Publication number: WO2008022486A1
Application number: PCT/CN2006/001723
Authority: WO
Inventors: Lei Chen; Jianyong Xu
Original assignee: Beijing North Elite Biotechnology Co., Ltd.
Priority date: 2006-07-17
Filing date: 2006-07-17
Publication date: 2008-02-28
Also published as: CN101490079A; CN101490079B

Abstract

Provided are a plant stress tolerance related isozyme protein, encoding gene and use thereof. The protein has the amino acid residue sequences selected from: 1) SEQ ID No:l and SEQ ID No:2 in the sequence list; 2) SEQ ID No:l or SEQ ID No:2 derivative protein with a sequence of SEQ ID No:l or SEQ ID NO:2 altered through substitution and/oτ deletion and/or addition of one or several amino acid residues and possessing stress tolerance ability. The protein and gene can promote plant growth and increase stress tolerance of plant.

Description

Plant growth and stress tolerance related isozymes and their coding genes and applications

The invention relates to an isozyme related to plant growth and stress tolerance and a coding gene thereof and application thereof in the field of plant biotechnology, in particular to two species derived from rice grass S _P arti

Anglica) leaf follicle H+-pyrophosphatase isozyme associated with plant growth and stress tolerance

(vacular H ⁺ — pyrophosphatase ) and its coding genes and applications.

Background technique

Plants in nature often suffer from stressful stresses such as drought, salinity and low temperatures, which inhibit their growth and development and even lead to plant death. Plant resistance to stress or tolerance is called stress tolerance and is the adaptation of plants to adverse environments during long-term evolution. For many years, people have studied the relationship between plants and stress from various angles. From the initial study of physiological phenomena to the study of ecology and genetics, to the study of physiology, biochemistry and metabolism, a wealth of information has been accumulated. Especially with the development of molecular biology, people can understand the tolerance mechanism of plants to stress at the molecular level of gene composition, expression regulation and signal transduction, and open up new ways to improve the resistance of plants to salt stress by genetic engineering. way. Due to the complexity of plant stress tolerance, it is very difficult to improve the tolerance of plants by traditional breeding methods. With the development of molecular biology, genetic engineering has improved the tolerance of plants and opened up new varieties of plant tolerance breeding. way.

Leaf bubbles account for 40% _99% of the total plant cell volume (Rea, P. A., et

Al. , Tonoplast Adenosine Triphosphate and Inorganic

Pyrophosphatase. In -.Methods Plant Biochem., pp. 385 - 405,

Academic Press Limited, London (1990) ) is a relatively independent chamber in the cell that is responsible for storing nutrients, maintaining the stability of the cytoplasmic pH, isolating toxic anions and cations in cells, maintaining cell turgor, and regulating cells. The second messenger Ca ion concentration in the plasma plays an important role in the physiological and biochemical processes of plant cells. The proton pump is positioned on the leaf vesicle to feed hydrogen protons into the leaf vesicles (Rea, PA, et al., Tonoplast Adenosine Triphosphate and inorganic Pyrophosphatase. In : Methods Plant Biochem., pp. 385-405, Academic Press Limited, London (1990)) The resulting difference in pH and potential between the inside and outside of the leaf follicle provides a major source of energy for the transport of ions inside and outside the leaf. There are two types of hydrogen proton pumps positioned in leaf follicles: vesicular H+- ATPase (vacular ATPase) and H+-pyrophosphatase (H ⁺ -PPase) (Davies, JM, et al., The Bioenergetics of Vacuolar H. sup. + Pumps. In : Plant Vacuole, pp. 340-363, Leigh, RA, Sanders, D. (eds.), Academic Press, San Diego (1997) ) . V-ATPase is made up of at least 26 Gene-encoded subunit composition (Sze H, Schumacher K,

Muller ML, Padmanaban S, Taiz L (2002) A simple nomenclature for a complex proton pump : VHA genes encode the vacuolar H-ATPase. Trends Plant Sci 7 : 157 - 161 ) , H+- PPase is encoded by a single gene, There are two H ⁺ - PPase genes in Arabidopsis: AVPl (V. Sarafian, Y. Kim, RJ Poole, PA Rea, Proc. Natl. Acad. Sci. USA 89, 1775 (1992) · ) and AVP2 (N Mitsuda, K. Enami, M. Nakata, K. Takeyasu, MH Sato, FEBS Lett. 488, 29 (2001) . ) , the former is located on the leaf membrane and the homology at the protein level is 35%. . Overexpression of the AVP1 gene can enhance the activity of various pump proteins on the leaf cells of transgenic plants, such as Ca/H ⁺ , Na/H ⁺ pumps, etc., thereby enhancing the drought tolerance and salt tolerance of transgenic plants.

(Gaxiola RA, Li J, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR (2001) Drought- and salt-tolerant plants result from overexpression of the AVPl H-pump. Proc Natl Acad Sci USA 98 : 11444 - 11449). At the same time, recent studies have shown that AVP1 is also localized on the cell membrane. Overexpression of AVP1 can increase the number of P-ATPase on the cell membrane, enhance the extracellular extracellularity, thereby increase the transport of cytokine to cells, and promote the development and growth of various organs of plants. (Science. 2005 Oct 7 : 310 (5745) : 121 - 5. Arabidopsis H+-PPase AVPl regulates auxin-mediated organ development. Li J, Yang H, Peer WA, Richter G, Blakeslee J, Bandyopadhyay A, Titapiwantakun B, Undurraga S, Khodakovskaya M, Richards EL, Krizek B, Murphy AS, Gilroy S, Gaxiola R.).

Invention disclosure

It is an object of the present invention to provide two isozyme proteins and their coding genes which are related to plant growth and stress tolerance.

There are two plant growth and stress tolerance related isozyme proteins provided by the present invention, named DMYVVP1 and DMYVVP2, which are derived from rice grass and are proteins having one of the following amino acid residue sequences:

1) SEQ ID No _: 1 in the sequence listing;

2) SEQ ID Na: 2 in the sequence listing;

3) Pass the amino acid residue sequence of SEQ ID Nfi: 1 in the sequence listing through one or several amino groups a protein that is substituted and/or deleted and/or added to an acid residue and is associated with plant growth and stress tolerance; 4) the amino acid residue sequence of SEQ ID No: 2 in the sequence listing is passed through one or several amino acid residues Proteins that are substituted and/or deleted and/or added and are associated with plant growth and stress tolerance.

Among them, the sequence 1 in the sequence listing consists of 764 amino acid residues. The amino acid residues 69 to 749 of the amino terminus of DMYVVP1 are the H+-pyrophosphatase domain.

Sequence 2 in the sequence listing consists of 772 amino acid residues. The amino acid residues 55 to 758 of the amino terminus of DMYVVP2 from Sequence 2 are H ⁺ -pyrophosphatase domains.

Substitutions and/or deletions and/or additions of the one or more amino acid residues refer to substitutions and/or deletions and/or additions of no more than ten amino acid residues.

The above plant growth and stress tolerance related isozyme protein encoding genes (dmyvvpl and dmyvvp2) are also within the scope of the present invention.

The above cDNA gene for plant growth and stress tolerance related isozyme protein may have one of the following nucleotide sequences:

1) the DNA sequence of SEQ ID Na: 3 in the Sequence Listing;

2) the DNA sequence of SEQ ID Na: 4 in the sequence listing;

3) a polynucleotide encoding a protein sequence of SEQ ID Na: 1 in the sequence listing;

4) a polynucleotide encoding a SEQ ID Na: 2 protein sequence in the sequence listing;

5) a nucleotide sequence which hybridizes under high stringency conditions to a DNA sequence defined by SEQ ID Na: 3 in the Sequence Listing;

6) A nucleotide sequence which hybridizes under high stringency conditions to a DNA sequence defined by SEQ ID No _: 4 in the Sequence Listing.

The above high stringency conditions may be to wash the membrane at 56 ° C in a solution of 1 X SSC, 0.1% SDS.

Wherein, SEQ ID No _: 3 in the sequence listing consists of 2603 deoxynucleotides, and the deoxynucleotides from positions 5' to 2620 are coding sequences.

Wherein, SEQ ID Na: 4 in the sequence listing consists of 2563 deoxynucleotides, and the deoxynucleotides from positions 5' to 41 are the coding sequence.

Expression vectors, cell lines, host bacteria and expression cassettes containing the gene of the present invention are all within the scope of the present invention.

Expression vectors containing dmyvvpl and dmy _VV p2, such as pC2300::dmyvvpl and pC2300::dmyvvp2 (physical maps as shown in Figures 1 and 2), can be obtained by existing methods of molecular biology. The expression cassette includes a plant promoter, dw, l and ?7 gene and a terminator. The plant promoter can be a constitutive, inducible or tissue specific promoter.

The invention

And when the gene is constructed into a plant expression vector,

Any one constitutive promoter, tissue-specific promoter or inducible promoter may be added before the transcription start nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, the vectors used can be processed, such as the addition of plant selectable markers (GUS gene, luciferase gene, etc.) or resistant antibiotic markers (Qing Damycin, kanamycin, etc.). The transformed plant host can be either a monocot or a dicot, wherein the monocot

105 can be turfgrass, wheat, barley, oats, rice or corn, etc. Dicotyledon can be horsebell

Potato, tobacco, cotton, lettuce, tomato, melon, cucumber, pea, oilseed, beet or sunflower.

Carrying the invention

The gene expression vector can be transformed into a plant cell or tissue by using conventional biological methods such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, Agrobacterium 110 mediated, and the transformed material is organized. Cultivate into plants.

The ^^? and ?7^ genes of the present invention can be widely used for cultivating growth-enhancing and resistance-resistant transgenic plants by functionally linking to a promoter having a specific function.

DRAWINGS

115 Figure 1 shows the physical map of dmyvvpl plant expression vector

Figure 2 shows the physical map of plant expression vector

Figure 3 is a PCR verification photo of transgenic pC2300: : dmyvvpl tobacco

Figure 4 is a PCR verification photo of transgenic pC2300: : dmyvvp2 tobacco

The best way to implement the invention

120 The experimental methods in the following examples are all conventional methods unless otherwise specified. The practice of the present invention uses conventional molecular biology, cell biology, and cloning techniques in the art, unless otherwise indicated. These techniques are well known to the skilled person and are explained in detail in the literature. See, for example, Sambrook and Russell "Molecular Cloning: A Laboratory Manual" (2001); Cloning: A Practical Approach, "Volumes I and 125 II (DN Glover, ed., 1985); Yan Wang Guanlin, Fang Hongwei, "Plant Genetic Engineering "The second edition, the second edition of 2002.

Example 1. Acquisition of DMYVVP1 and DMYVVP2 and their coding genes

Millet leaves prepared by liquid nitrogen - 70 ° C, add lml per 0. lg of plant leaves TRIZOL RNA Extract (Dingguo Biotech Co., Ltd.), and total RNA extraction was performed according to the protocol provided by the supplier. The obtained total RNA was digested with DNase (Prmega, America) to remove residual DNA, and the concentration of total RNA in the sample was measured by a spectrophotometer (Eppendorf, Germany). I g total RNA was reverse-transcribed by the reverse transcription kit (Bao Bioengineering (Dalian) Co., Ltd.) according to the kit method, and the obtained cDNA fragment was used as a template for PCR amplification reaction, and the cDNA fragment of DMYWP1 was amplified. . Primers were designed based on the known conserved regions of H ⁺ —pyrophosphatase protein. Primers were: F1 : 5 ' ― (A/G) GC (A/T/G) GC ( C/T) GATGT (G/T/ C) GGTGC—3 '; R1 : 5 ' I—(A/T/G/C) CC (T/A) CC (A/G) GC (A/G) TT

(A/G) TCACT- - 3 '. The 25 ul PCR reaction system was: 1 gc awake, 1. 5 mM MgCl ₂ , 20 mM Tris-HCl (pH 8.4), 50 mM I (C1, 0.8 mM dNTP mixture, 1 μΜ primer Fl and 1 μΜ primer Rl, And 1 unit of Taq polymerase (Shanghai Shenneng Gaming Biotechnology Co., Ltd.) PCR cycle was carried out in a PCR-thermal cycler (Eppendorf, Germany) according to the following protocol: pre-denaturation at 94 °C for 5 minutes; then 94 Ό 1 minute , 55 ° C 1 minute, 72 ° C 1 minute, a total of 30 cycles; the last 72 ° C 10 minutes. The amplified about 780 bp DNA fragments using the gel recovery kit (Beijing Tianwei Times Technology Co., Ltd.) The fragment was recovered according to the protocol provided by the kit, and the fragment was cloned by the pGEM-Teasy vector system (Promega, USA) according to the protocol provided by the kit, and sequenced (Sanbo Yuanzhi Biotechnology Co., Ltd.) to obtain two highly homologous sequences. Sequence analysis indicated that these two sequences are conserved regions of the H+-pyrophosphatase protein gene, and it is preliminarily concluded that there are at least two H+-pyrophosphatase in ricegrass. According to the two conserved segments, 5 ' RACE and

3 ' RACE specific primers, 1w ng total RNA for 5' RACE and 3' RACE. The RACE program was carried out according to the instructions of the 5' RACE and 3' RACE kits provided by Biosciences (Dalian) Co., Ltd. The primer for 5' RACE of DMYVVP1 is: Phosphorylation RT Primer: 5, -GCAGCGCAGGAAGA-3, , S2: 5 ' - CTCTCAACTTTGCCAACAAGATCA - 3, , S1 : 5, A GATCATCCTCGGGGATGTTCCT -3, , A1 : 5, - CGTTGGTGACAATGTCGGTGACA - 3, , A2 : 5' ― TGTCGGTGACATTGCTGG - 3 ' . The reverse transcription reaction was carried out with phosphorylated RT primers, and the first round of amplification was carried out with S1 and A1 as primers, and then the first round of PCR products diluted 100 times was used as a template, and S2 and A2 were used as primers for the second round. PCR amplification. The 25ul PCR reaction system is: 1 g cDNA, 1. 5 mM MgCl ₂ , 20 mM Tris-HCl (pH 8.4), 50 mM KC1, 0.8 mM dNTP mixture, 1 μΜ forward and reverse primers, and 1 Unit of Pfu enzyme (Shanghai Shenneng Gaming Biotechnology Co., Ltd.). PCR cycles were performed in a PCR-thermal cycler (Eppendorf, Germany) according to the following protocol. Among them, the first round of PCR reaction conditions: 94 Ό pre-denaturation for 5 minutes; another 94 Ό 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute, A total of 30 cycles; the last 72 Ό 10 minutes. The second round of PCR reaction conditions: 94 Ό pre-denaturation for 5 minutes; another 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 1 minute and 30 seconds for a total of 30 cycles; finally 72 ° C for 10 minutes.

The 5' end of the 3' RACE of YVVP1 is: 5, - CTTGTTGGCAAAGTTGAGAGGAA - 3, and the 3 ' primer is: 5, -CTCGAGTCTAGATTTTTTTTTTTTTTTTTT- 3, . Reaction conditions: pre-denaturation at 94 ° C for 5 minutes; further at 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 1 minute and 30 seconds for a total of 30 cycles; and finally 72 ° C for 10 minutes.

The obtained 5, -RACE and 3, - RACE products were cloned and sequenced, and the primers were synthesized. The total RNA of 1μ _§ rice grass leaves was reversed by the kit (Bao Bioengineering (Dalian) Co., Ltd.) according to the kit method. The recorded cDNA was used as a template, and the cDNA of the DMYVVP1 gene was cloned. The synthesized primer was 5' primer F2: 5'-gatatcGGTTAAGGTTCTGGTGGCCG (with a coR V site at the end), 3' primer R2: 5'-ctcgagGGCAGGAGTTATTTGTGATG (with a Xhol site at the end); 25ul PCR reaction system: 1 g cDNA , 1. 5 niM MgCl ₂ , 20 raM Tris-HCl (pH 8.4), 50 mM KC1, 0. 8raM dNTP mixture, 1 μΜ F2 and 1 μΜ R2, and 1 unit of Pfu enzyme (Shanghai Shenneng Gaming Creature) Technology company). PCR cycles were performed in a PCR-thermal cycler (Eppendorf, Germany) for amplification according to the following protocol

cDNA of DMYVVP1 gene: pre-denaturation at 94 °C for 5 minutes; further 1 minute at 94 °C, 1 minute at 50 °C, 1 minute and 30 seconds at 72 °C for 30 cycles; last 72 minutes for 10 minutes. The amplified fragment was recovered by a gel recovery kit (Beijing Tianwei Times Technology Co., Ltd.) according to the protocol provided by the kit, inserted into pGEM-T vector (promega), and sequenced (Sanbo Yuanzhi Biotechnology Co., Ltd.). The sequencing results showed that the fragment was 2603 bp in size and had the nucleotide sequence of sequence 2 in the sequence listing, which was named DMYVVP1. Sequence analysis indicated that the cDNA contained amino acids of sequence 1 from the 5' 26th to 2320 deoxynucleotides. Residue sequence of DMYVVP1. The amino acid residues 69 to 749 of the amino terminus of DMYVVP1 from sequence 1 are H ⁺ -pyrophosphatase domains. The plasmid containing the gene was named PGEM:: drayvvpl o

The primer for 5' RACE of DMYVVP2 is: Phosphorylated RT Primer: 5, - AGCAAGGCTGAAGC - 3, , S2 : 5 ' - AACGACAAGAGCAGCACA -3 , , S1 : 5 ' - AAAGGGTGGTTATCAAGC -3, , A1 : 5, - AGAGCCAGCCCTCAAGAA - 3 , , A2 : 5 ' A CTGCCGTGATGACTGTTG - 3 '. The reverse transcription reaction was carried out with phosphorylated RT primers, and the first round of amplification was carried out with S1 and M as primers, and then the first round of PCR products diluted 100 times was used as a template, and S2 and A2 were used as primers for the second round. PCR amplification. The 25ul PCR reaction system is: 1 g cDNA, 1. 5 mM MgCl ₂ , 20 mM Tris-HC1 (ρΗ8·4), 50 mM KC1, 0. 8raM dNTP mixture, 1 μΜ forward and reverse primers, and 1 unit of Pfu enzyme (Shanghai Shenneng Gaming Biotechnology Co., Ltd.). Follow the protocol below in a PCR-thermal cycler (Eppendorf, Germany)

195 Perform PCR cycle. Among them, the first round of PCR reaction conditions: 94 Ό pre-denaturation for 5 minutes; another 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 1 minute, a total of 30 cycles; the last 72 ° C for 10 minutes. The second round of PCR reaction conditions: 94 Ό pre-denaturation for 5 minutes; another 94 ° C for 1 minute, 5 (TC 1 minute, 72 ° C 1 minute 30 seconds, a total of 30 cycles; finally 72 ° C for 10 minutes.

The 5' end of the 3' RACE of DMYVVP2 is: 5, - TGAAATAGAGCCAGCCCTCA

200 - 3' , 3, the end primers are: 5' -CTCGAGTCTAGATTTTTTTTTTTTTTTTTTTT- 3'.

Reaction conditions: 94 Ό pre-denaturation for 5 minutes; further at 94 ° C for 1 minute, 56 ° C for 1 minute, 72 ° C for 1 minute and 30 seconds for a total of 30 cycles; and finally 72 ° C for 10 minutes.

The obtained 5'-RACE and 3'-RACE products were cloned and sequenced, and the primers were synthesized. The total RNA of the leaves of the rice grass was reverse-transcribed with a kit (Bao Bioengineering (Dalian) Co., Ltd.).

The cDNA obtained by reverse transcription of the method of 205 was used as a template to clone the cDNA of the DMYVVP2 gene. The synthesized primer was 5' primer F3: 5'-gatatcAAAGACCCAAGCGCTTCG (end with ^oR V site), 3, primer R3: 5 '-ctc gagGGATTCGTCATCATAATAAATTC (end with Xhol site); 25ul PCR reaction system: 1 Μ§ cDNA, 1. 5 mM MgCl ₂ , 20 mM Tris-HCl (pH 8.4), 50 mM KC1, 0.8 mM dNTP mixture, 1 μΜ F2 and 1 μΜ

210 R2, and 1 unit of Pfu enzyme (Shanghai Shenneng Gaming Biotechnology Co., Ltd.). PCR cycles were performed in a PCR-thermal cycler (Eppendorf, Germany) for amplification according to the following protocol

cDNA of DMYVVP1 gene: pre-denaturation at 94 °C for 5 minutes; further 94 Ό 1 minute, 50 Ό 1 minute, 72 ° C 1 minute 30 seconds for 30 cycles; finally 72 ° C for 10 minutes. The amplified fragment is obtained by the gel recovery kit (Beijing Tianwei Times Technology Co., Ltd.) according to the kit.

215 The fragment was recovered, inserted into pGEM-T vector (promega), sequenced (Sanbo Yuanzhi Biotechnology Co., Ltd.), and the sequencing result showed that the fragment was 2563 bp in size, and the nucleotide sequence of sequence 2 in the sequence listing was named DMYVVP2; Analysis showed that the deoxynucleotide from position 5' to position 4359 of the cDNA encodes DMYVVP2 having the amino acid residue sequence of sequence 1. The amino acid residues 55 to 758 of the amino terminus of DMYVVP2 are the H+-pyrophosphatase domain.

220 Example 2. Construction of DMYVVP1 and DMYVVP2 plant expression vectors and growth of transgenic tobacco and stress tolerance experiment

Using CTAB method (Steiner JJ, Poklemba CJ, Fjellstrom RG, Elliott LF., A rapid one-tube genomic DNA extraction process for PCR and RAPD analyses. Nucleic Acids Res. 1995 Jul 11 ; 23 (13) : 2569- 70.) Extraction of rice genomic DNA, the specific method is as follows: Take rice leaves, grind with liquid nitrogen, add 5 ml of 68 Ό preheated CTAB solution (with 10 mM mercaptoethanol before use) per gram of plant material, 60 Ό Heat for 30 minutes. Then, an equal volume of chloroform:isoamyl alcohol was added, and the mixture was centrifuged at 15,000 g for 15 minutes, and the supernatant was taken. Add 2/3 volume of isopropanol to the supernatant, mix well, stir the DNA filament into the new tube with the needle tip, add 75% ethanol, wash the pellet and centrifuge the tube wall, then add ethanol. Drain dry, air dry the remaining ethanol, and add TE to dissolve the DNA. And stored at - 20 ° C. The promoter sequence of the rice act in gene was amplified by PCR. The 25 ul PCR reaction system contains 100 ng of genomic DNA, 1. 5 mM MgCl ₂ , 20 mM Tri s-HC1 (pH 8.4), 50 mM KC1, 0.8 mM dNTP mixture, 1 μΜ Actin-S and 1 μΜ act in - A, 1U Taq polymerase (Shanghai Shenyou Biotechnology Co., Ltd.). According to the following scheme in PCR-thermal cycler

PCR cycle (Eppendorf, Germany): first 94 ° C for 5 minutes; then 94 ° C for 1 minute, 54 ° C for 1 minute, 72 ° C for 1 minute, a total of 30 cycles; the last 72 ° C for 10 minutes. Actin-S: 5, - gat at cTCTTCTACCTACAAAAAAGCTCC-3 ' (end with ^ d? V site), actin- A : 5, - gatatcGTCATTCATATGCTTGAG - 3, (with Eco V site at the end). The amplified DNA fragment of about 1398 bp was recovered by a gel recovery kit (Beijing Tianwei Times Technology Co., Ltd.) according to the protocol provided by the kit, inserted into the pGEM-T vector (promega), and sequenced (Sanbo Yuanzhi) Biotech company), obtained plasmid pGEM :: Pact in.

The 3' end sequence of the rice act in gene was amplified by PCR. The 25ul PCR reaction system contains 100 ng of genomic blue, 1.5 mM MgCl ₂ , 20 mM Tri s-HC1 (pH 8, 4), 50 mM KC1, 0.8 mM dNTP mixture, 1 μΜ Tact in-S and 1 μΜ Tact in-A, 1U Taq polymerase (Shanghai Shenyou Biotechnology Co., Ltd.). The PCR cycle was carried out in a PCR-thermal cycler (Eppendorf, Germany) according to the following protocol: first 94 ° C for 5 minutes; then 94 ° C for 1 minute, 54 ° C for 1 minute, 72 ° C for 1 minute, a total of 30 cycles The last 72 Ό 10 minutes.

Tactin- S : 5 ' -aaagt c gacCTTCGGACCCAAGAATGCTA -3, (with a 63⁄4 I site at the end), Tactin- A : 5' -aaagtcgactctagaCGAGCTCGAATTCGTAATCA-3 '

(with Sal I and Xba I sites at the end). The amplified DNA fragment of about 1430 bp in length was recovered by a gel recovery kit (Beijing Tianwei Times Technology Co., Ltd.) according to the protocol provided by the kit, inserted into pGEM-T vector (promega), and sequenced (Sanbo Yuanzhi) Biotech company), obtained plasmid pGEM : : Tactin.

The plasmid pGEM::Tact in was digested with Ssd I and the fragment containing the 3' end of the rice actin gene was inserted into the Xho I site of pGEM::dmyvvpl to obtain plasmid pGEM::dmyvvpl- Tactir

The plasmid pGEM::Pactin was digested with the Eco V enzyme and the promoter sequence containing the rice actin gene was inserted into the ed? V site of pGEM : : dmyvvpl- Tactin to obtain the plasmid pGEM : i P- dmyvvpl - T.

The pdexM::P-dmyvvpl-T was digested with S&1 I and Xba I, and the obtained dmyvvpl expression cassette was cloned into the 63⁄4 I and J0a I recognition sites of pCAMBIA2300 (GAMBIA) to obtain the plant expression vector of dmyvvpl. pC2300 _: : dmyvvpl , the plasmid map is shown in Figure 1. In this vector, dmyvvpl is regulated by the rice actin promoter. The pC2300::dmyvvpl was transformed into Agrobacterium tumefaciens LBA4404 to obtain an Agrobacterium strain containing pC2300::dmyvvpl, designated as TpCdvpl, and the plasmid pCAMBIA2300 was transformed into Agrobacterium tumefaciens LBA4404 to obtain a strain containing PCAMBIA2300 empty vector, which was named TpCAMBIA2300.

Similarly, the plasmid pGEM: : Tact in was digested with the S&1 I enzyme to insert a fragment containing the 3' end sequence of the rice actin gene into the Xho I site of pGEM::dmyvvp2 to obtain the plasmid pGEM::dmyvvp2-Tactin.

The plasmid pGEM:: Pactin was inserted into the coR V site of pGEM::dmyvvp2 -Tact in with the coR V enzyme and the plasmid pGEM :: P - dmyvvp2-T was obtained.

The pGEM::P-dmy ννρ2-Τ was digested with S&l I and Xba I, and the resulting d/nyvvp2 expression cassette was cloned into the S&1 I and Xba I enzyme recognition sites of pCAMBIA2300 (CAMBIA) to obtain dmywp2. The plant expression vector pC2300::dmyvvp2, the plasmid map is shown in Figure 2, in which dmyvvp2 is regulated by the rice actin promoter. The pC2300 _:: dmyvvp2 was transformed into Agrobacterium tumefaciens LBA4404 to obtain an Agrobacterium strain containing pC2300::dmyvvp2, designated as TpCdvp2, and the plasmid pCAMBIA2300 was transformed into Agrobacterium tumefaciens LBA4404 to obtain a strain containing pCAMBIA2300 empty vector, which was designated as TpCAMBIA2300.

The tobacco sterile seedling leaves were cut into four-sided leaf discs of about 1 cm with a sterile blade, and immersed in TpCdvpl, TpCdvp2 and TpCAMBIA2300 (transfer empty vector control) with a value of 0. D. for 10 minutes, respectively. Into the co-cultivation medium (MS basic medium plus 6-benzylaminopurine 1. 0 mg / L, indole acetic acid 0.1 rag / L, sucrose 30 g / L, pH 5. 2), dark at 25 ° C After 3 days of culture, transfer to subculture medium (MS basic medium plus 6-benzylaminopurine 1.0 rag/L, indole acetic acid 0.1 m _g / L, carbenicillin 250 mg/L, sucrose 30 g / L, pH 5. 8 ) medium light culture for three days, transferred to the screening medium (MS basic medium plus 6-benzylaminopurine 1.0 rag / L, indole acetic acid 0.1 mg / L, carboxy Benzicillin 250 mg/L, kanamycin 75 mg/L, Sucrose 30 g / L, pH 5. 8 ); continue to culture for 7 days, then transferred to regeneration medium (MS basic culture)

290 Nutrient plus 6-benzylaminopurine 1. 0 mg/L, carbenicillin 250 mg/L, kanamycin

75 mg / L, sucrose 30 g / L, pH 5. 8). When the buds of the leaves grow to 2 - 3 cm, the regenerated shoots are cut with a scalpel and transferred to rooting medium (MS basic medium plus kanamycin 50 mg/L, sucrose 20 g/L, In the pH 5. 8 . ) culture for 20 days, a total of 21 transgenic PC2300 were obtained: : dmywpl plants, 24 transgenic pC2300: : dmyvvp2 plants and 13 transgenic plants

295 pCAMBIA2300 plants.

Using the CTAB method (Steiner JJ, Poklemba CJ, Fjellstrom RG, Elliott LF., A rapid one-tube genomic DNA extraction process for PCR and RAPD analyses. Nucleic Acids Res. 1995 Jul 11 ; 23 (13): 2569- 70. ) Extracted separately to pC2300: : dmyvvpl, pC2300: : dmyvvpl and pCAMBIA2300

300 Tobacco genomic DNA. The dmyvvpl gene was amplified by PCR with primers F2 and R4: TGCAATACCAGCGGTCATCA, and the dmyvvp2 gene was amplified by PCR with primers F3 and R5: CAGCAATATCTCCAACATTATCACC. The reaction system is 25 ul, containing 100 n _{g of} genomic DNA, 1.5 mM MgCl ₂ , 20 mM Tris-HCl (pH 8.4), 50 mM KC1, 0.8 mM dNTP mixture, 1 μΜ F2 and 1 μΜ R2,

305 1U Taq polymerase (Shanghai Shenyou Biotechnology Co., Ltd.). The PCR cycle was carried out in a PCR-thermal cycler (Eppendorf, Germany) according to the following protocol: first 94 ° C for 5 minutes; then 94 Ό 1 minute, 58 Ό 1 minute, 72 Ό 1 minute, a total of 30 cycles; finally 72 ° C 10 minute. The amplified product was subjected to electrophoresis, and the results are shown in Fig. 3 and Fig. 4. The _P C2300: : dmyvvpl tobacco amplification product has a clear band at an estimated 1150 bp, and the pC2300: : ctaiyvvp2 tobacco expansion

The 310 product had a clear band at the expected approximately 900 bp, whereas the transfected pCAMBIA2300 (empty vector control) tobacco had no PCR product bands. In Figure 3, lane 1 is the PCR amplification product of transgenic _P CAMBIA 2300 tobacco; lane 2 is the PCR amplification product of pC2300: : dmyvvpl plasmid; lane 3 is the PCR amplification product of transgenic pC2300: : dmyvvpl tobacco; lane M is lkbDNA Molecular weight standard.

315 In Figure 4, lane 1 is a PCR amplification product of transgenic pCAMBIA2300 tobacco; lane 2 is

PC2300: : PCR amplification product of dmyvvp2 plasmid; Lane 3 is a PCR amplification product of transgenic pC2300: :drayvvp2 tobacco; Lane M is the molecular weight standard of lkbDNA.

Incubate for 30 days in rooting medium (MS basic medium plus kanamycin 50 mg/L, sucrose 20 g/L, pH 5. 8). Turn the pC2300: : dmyvvpl tobacco and turn pC2300: :

320 dmyvvp2 tobacco compared to transgenic pCAMBIA2300 (empty vector control) tobacco, transgenic _P C2300: : Dmyvvpl and pC2300: : dmyvvp2 The main roots of tobacco and the number of roots are large, the main root is long, and the thousand matter of the plant is heavier.

Transfer PC2300: : dmyvvpl > pC2300: : dmyvvp2 Tobacco and transfer to pCAMBIA2300 (empty vector control) Tobacco was placed in a nutrient solution and watered 10 times a day with 10 times diluted MS nutrient solution. After 4 weeks, the water was stopped and observed. Transgenic pC2300: : dmyvvpK pC2300: : dmy _VV p2 Tobacco compared to pCAMBIA2300 Tobacco was slowed down, reconstituted after 10 days, transferred to PC2300: : dmyvvpl > pC2300: : dmyvvp2 Tobacco recovered faster than pCAMBIA2300. This experiment shows that transgenic PC2300: : dmyvvpU pC2300: : dmyvvp2 tobacco has stronger drought tolerance.

330 Industrial Applications

Experiments have shown that the protein of the present invention and its encoding gene can enhance plant growth and enhance plant tolerance to stress, especially drought stress. This gene will be widely used to cultivate resistant plants, which has far-reaching theoretical significance and wide practical significance.

Claims

Claim 335

A plant growth and stress tolerance related isozyme protein which is a protein having one of the following amino acid residue sequences:

1) SEQ ID No _: 1 in the sequence listing;

2) SEQ ID Na: 2 in the sequence listing;

340 3 ) a protein which has been subjected to substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid residue sequence of SEQ ID No _: 1 in the sequence listing and which is related to plant growth and stress tolerance;

4) A protein in which the amino acid residue sequence of SEQ ID 0: 2 in the Sequence Listing is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and is associated with plant growth and stress tolerance.

The protein according to claim 1, wherein the protein has the amino acid residue sequence of SEQ ID Nfi _: 1 in the sequence 345.

The protein according to claim 1, wherein the protein has an amino acid residue sequence of SEQ ID a: 2 in the Sequence Listing.

The coding gene for plant growth and stress tolerance related isozyme protein according to claim 1 to 3.

The gene according to claim 4, wherein the plant growth and stress tolerance related isozyme protein cDNA is one of the following nucleotide sequences:

1) the DNA sequence of SEQ ID Na: 3 in the Sequence Listing;

2) the DNA sequence of SEQ ID No _: 4 in the Sequence Listing;

3) a polynucleotide encoding a protein sequence of SEQ ID No _: 1 in the Sequence Listing; 355 4) a polynucleotide encoding a SEQ ID Ns _: 2 protein sequence in the Sequence Listing;

5) a nucleotide sequence which hybridizes under high stringency conditions to a DNA sequence defined by SEQ ID No _: 3 in the Sequence Listing;

360. An expression vector comprising the gene of claim 4 or 5.

7. A cell line comprising the gene of claim 4 or 5.

8. A host strain comprising the gene of claim 4 or 5.

9. An expression cassette comprising the gene of claim 4 or 5.

The use of the plant growth and stress tolerance related isozyme protein according to claim 1 and the coding 365 gene for cultivating a stress-tolerant plant.

The use according to claim 10, characterized in that the stress tolerance is drought tolerance.