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Gossypium hirsutum serine/threonine protein phosphatase GhTOPP6 as well as coding gene and application thereof

A gene and protein technology, applied in the field of cotton silk/threonine protein phosphatase GhTOPP6 and its coding gene and application, can solve the problems that there are no research reports on PP1 members

Active Publication Date: 2020-12-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PP1 is an important class of phosphatases in the PPP protein phosphatase family. In Arabidopsis, PP1 has 9 members, and there is no research report on PP1 members in cotton.

Method used

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  • Gossypium hirsutum serine/threonine protein phosphatase GhTOPP6 as well as coding gene and application thereof
  • Gossypium hirsutum serine/threonine protein phosphatase GhTOPP6 as well as coding gene and application thereof
  • Gossypium hirsutum serine/threonine protein phosphatase GhTOPP6 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1. Discovery, cloning and localization of GhTOPP6 protein and its coding gene

[0080] 1. Discovery of GhTOPP6 protein and its coding gene

[0081] Screening of salt-related genes in the VIGS cDNA library (using VIGS technology to study the function of cotton stress resistance genes, Li Fangjun, "China Agricultural University", 2014), using the cotton database to search from the cotton variety "Guoxin 3" (Guoxin 3 Insect-resistant cotton cultivation technology, Feng Sulian, "Hebei Agriculture", 2011, 06) obtained a new protein and named it GhTOPP6 protein. The amino acid sequence of the GhTOPP6 protein is shown in SEQ ID No.1, consisting of 323 Composition of amino acid residues; the gene encoding the GhTOPP6 protein is named GhTOPP6 gene, and the open reading frame of the GhTOPP6 gene is shown in SEQ ID No.2, consisting of 972 nucleotides.

[0082] 2. Cloning of GhTOPP6 gene

[0083] Cotton leaf and root RNA was extracted using the Aidelai kit (the kit was pu...

Embodiment 2

[0104] Example 2, VIGS silenced plant stress phenotype

[0105] 1. Construction of VIGS-GhTOPP6 silencing vector

[0106] 1. Extract the total RNA from the leaves of the cotton variety "Guoxin 3" and reverse transcribe it into cDNA.

[0107] 2, with the cDNA that step 1 obtains as template, carry out PCR amplification with the primer pair that F1 and R1 form, obtain PCR amplification product ( Figure 5 ).

[0108] F1: 5'-G GAATTC ATGGAAACTAGGGTTCTT-3' (underlined is the restriction site of EcoRI)

[0109] R1:5'-CG GGATCC AGGAGAAGACATATGGTT-3' (the restriction site of BamHI is underlined)

[0110] 3. The PCR amplification product obtained in step 2 was double-digested with restriction endonucleases EcoRI and BamHI, and the digested product was recovered.

[0111] 4. Use restriction endonucleases EcoRI and BamHI to double digest the pYL156 (pTRV2:RNA2) vector (recorded in the non-patent literature "Gao X, 2013, Functional genomic analysis of cotton genes with agrobacteriu...

Embodiment 3

[0129] Embodiment 3, the acquisition of transgenic plants

[0130] 1. The 35S::GhTOPP6-GFP recombinant vector obtained in Step 4 of Example 1 was introduced into Agrobacterium strain GV3101 to obtain recombinant Agrobacterium.

[0131] 2. The recombinant Agrobacterium obtained in step 1 was shaken in LB liquid medium (50 μg / mL kanamycin, 25 μg / mL gentamycin) for 24 hours at 28° C., centrifuged at 4000 rpm for 10 minutes to collect the recombinant Agrobacterium and resuspended ( Resuspension: 50mM MES pH5.6, 5% sucrose, solvent is water), adjust the concentration of resuspension to OD 600 =0.8, add silwetL-77 (500μl / L, soak the inflorescence of Arabidopsis thaliana with the bacterial suspension, and then wrap the Arabidopsis thaliana with a black plastic bag to keep the humidity. Remove the plastic bag, restore the light, cultivate the plants according to the conventional method until they are firm, and harvest mature T. 0 generation seeds.

[0132] 3. Cultivate T in 1 / 2MS m...

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Abstract

The invention discloses gossypium hirsutum serine / threonine protein phosphatase GhTOPP6 as well as a coding gene and application thereof. The invention firstly discloses an application of any one of the following proteins to regulation and control of plant stress resistance: A1) a protein with an amino acid sequence of SEQ ID No. 1, A2) a fusion protein obtained by connecting a tag to the N terminal or / and C terminal of the amino acid sequence shown as SEQ ID No.1, and A3) a protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.1, has 90% or above of identity with the protein shown in A1) and has the same function as the protein shown in A1). The invention further discloses a method for cultivating a transgenic plant with enhanced stress resistance. The gossypium hirsutum GhTOPP6 gene can improve the stress resistance of plants, and lays a good molecular foundation for effectively improvingthe salt tolerance, drought resistance and ABA stress resistance of the plants.

Description

technical field [0001] The invention relates to the field of plant genetic engineering. It specifically relates to cotton silk / threonine protein phosphatase GhTOPP6 and its coding gene and application. Background technique [0002] Saline-alkali land is distributed in arid and coastal areas of all continents in the world. About 20% of the world's arable land is threatened by salt damage. Cotton is a pioneer crop planted in saline-alkali land. With the reduction of arable land, cotton planting gradually concentrates on saline-alkali land, but cotton is not a halophyte. , its salt tolerance is limited, and with the increase of soil salinity, cotton will also suffer from salt stress, and the cotton yield and quality problems in saline-alkaline cotton fields cannot be ignored. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/82A01H5/00A01H6/60A01H6/20
CPCC12N9/12C12N15/8218C12N15/8273C12N15/8293C12Y207/11
Inventor 李召虎李芳军周琳田晓莉
Owner CHINA AGRI UNIV
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