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Cotton protein tyrosine phosphatase GhPTP1 as well as encoding gene and application thereof

A coding gene and coding technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as backwardness and not many

Active Publication Date: 2017-06-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the current cotton gene cloning work has achieved certain results, the gene cloning work of cotton is far behind rice, corn, wheat and other food crops, especially the research on the mechanism of cotton salt tolerance, especially at the molecular level not many

Method used

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  • Cotton protein tyrosine phosphatase GhPTP1 as well as encoding gene and application thereof
  • Cotton protein tyrosine phosphatase GhPTP1 as well as encoding gene and application thereof
  • Cotton protein tyrosine phosphatase GhPTP1 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Discovery, cloning and localization of GhPTP1 protein and its coding gene

[0058] 1. Cloning of GhPTP1 gene

[0059] 1. Design of primers

[0060] According to the GhPTP1 gene sequence obtained from the cotton database, two specific primers were designed: upstream primer: ATGAAGATCGAGGATTTGG and downstream primer: GCAAGCACGTGCTTCA.

[0061] 2. Extraction of RNA

[0062] The Huayueyang kit was used to extract RNA from the leaves of cotton (cotton variety "Guoxin 3") (the kit was purchased from Beijing Jiukangyuan Biotechnology Co., Ltd., and the extraction was performed according to the instructions provided).

[0063] 3. Acquisition of cDNA

[0064] Using the RNA obtained in step 2 as a template, the first-strand cDNA was synthesized with an M-MLV reverse transcription kit (purchased from Promega, operated according to the kit instructions).

[0065] 4. PCR amplification

[0066] Use the cDNA obtained in step 3 as a template, and use the primers designe...

Embodiment 2

[0083] Example 2, the acquisition of GhPTP1 silenced plants and their salt and drought tolerant phenotypes

[0084] 1. Construction of VIGS-GhPTP1 silencing vector

[0085] 1. Extract the total RNA from the leaves of the cotton variety "Guoxin 3" and reverse transcribe it into cDNA.

[0086] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0087] F1: 5’-C TCTAGA ATGAAGATCGAGGATTTGGG-3';

[0088] R1:5'-G GGTACC GCAAGCACGTGCTTCATGAA-3'.

[0089] 3. Digest the PCR amplification product obtained in step 2 with restriction endonucleases XbaI and Kpn1, and recover the digested product.

[0090] 4. Digest the pTRV-RNA2 vector with restriction endonucleases XbaI and Kpn1 to recover the vector skeleton.

[0091] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain recombinant plasmid pTRV2-GhPTP1.

[0092] The recombinant plasmid pTRV2...

Embodiment 3

[0127] Example 3, Acquisition of Transgenic GhPTP1 Arabidopsis and Stress Resistance Analysis

[0128] 1. Construction of recombinant plasmids

[0129] 1. Extract the total RNA from the leaves of the cotton variety "Guoxin 3" and reverse transcribe it into cDNA.

[0130] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0131] Forward primer: F1:5'-C TCTAGA ATGAAGATCGAGGATTTGGG-3';

[0132] Reverse primer: R1:5'-G GGTACC GCAAGCACGTGCTTCATGAA-3'.

[0133] 3. Digest the PCR amplification product obtained in step 2 with restriction endonucleases XbaI and Kpn1, and recover the digested product.

[0134] 4. Digest the Super1300-GFP vector with restriction endonucleases XbaI and Kpn1 to recover the vector skeleton.

[0135] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant plasmid Super1300-GFP-GhPTP1.

[0136] The...

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Abstract

The invention discloses cotton protein tyrosine phosphatase GhPTP1 as well as an encoding gene and application thereof. The cotton protein tyrosine phosphatase GhPTP1 is a protein of (a), (b) and (c), wherein (a) an amino acid sequence is the protein as shown in SEQ ID NO: 2 in a sequence table; (b) a fusion protein is obtained by linking a label with the N-terminus and / or the C-terminus of the protein as shown in the SEQ ID NO: 2 in the sequence table; (c) a protein with the same function is obtained by carrying out substitution and / or deletion and / or addition on the amino acid sequence as shown in the SEQ ID NO: 2 in the sequence table by using one or several amino acid residues. Experiments prove that the GhPTP1 protein can reduce the stress resistance of plants, especially reduces the salt resistance, drought resistance and osmotic stress resistance of arabidopsis, and improves the sensitivity to ABA, thus laying a good molecular foundation for effectively improving the salt tolerance and drought resistance of the plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to cotton tyrosine protein phosphatase GhPTP1 and its coding gene and application. Background technique [0002] Cotton is the most important natural fiber economic crop in the world, and occupies a very important position in my country's national economy. At present, drought and salinity are one of the serious problems facing the world, and they are also the main factors restricting my country's agricultural production. Over the years, people have used traditional methods such as conventional breeding to solve the problems of drought and salinity in cotton varieties, but it often costs a lot of money, a lot of work, and the selection efficiency is not high. With the continuous development of molecular biology, it is becoming possible to cultivate drought-resistant and salt-tolerant cotton varieties by means of biotechnology. Although the current cotton gene cloning work h...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/82A01H5/00
CPCC07K2319/60C12N9/16C12N15/8271C12N15/8273C12N15/8293
Inventor 李召虎穆春张明才田晓莉李茂营杜明伟
Owner CHINA AGRI UNIV
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