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Phosphorylated protein kinase SAPK10 mutant and method thereof

A phosphorylated protein and mutant technology, applied in the field of genetic engineering and crop genetic improvement

Active Publication Date: 2018-08-31
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no related reports on the phosphorylation modification site of SAPK10, a member of rice SnRK2 kinase family gene, and its transgenic effect

Method used

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  • Phosphorylated protein kinase SAPK10 mutant and method thereof
  • Phosphorylated protein kinase SAPK10 mutant and method thereof
  • Phosphorylated protein kinase SAPK10 mutant and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1SAPK10 site-directed mutation

[0028] 1. Search for possible phosphorylation sites: Download 10 SnRK2 family members from Arabidopsis thaliana and 10 rice homologous proteins from the rice genome annotation database (http: / / rice.plantbiology.msu.edu / ) for sequence alignment Comparison( figure 1 ), it was found that the 177th serine in the SAPK10 protein sequence (SEQ ID NO: 1) is the most likely homologous site with the 175th serine in Arabidopsis OST1.

[0029] 2. Primer design for site-directed mutagenesis: Due to the different codon preferences of proteins of different species, the EditSeq software of DNASTAR Company was used to count the codons encoding alanine in SAPK10, and found that GCC in SAPK10 is the main alanine codon. It was therefore chosen to mutate SAPK10Ser-177 to GCC(Ala). By designing primers with mutated bases, the primer sequences are as follows:

[0030] Table 1 Primers for point mutation of SAPK10 protein Ser

[0031]

[0032] ...

Embodiment 2

[0053] Example 2 Vector Construction and Identification of Transgenic Positive Strains

[0054] 1. Construction of the binary vector: the mutant SAPK10 amplified in Example 1 S177A After the gene product is recovered, it is digested by KpnI (the restriction site is GAATCC) and BclI (the homologous enzyme of BamHI, the restriction site is TGATCA), and then ligated into PU1301 ( figure 2 ), to obtain PU1301-Ubiquitin-SAPK10 S177A The overexpression vector was verified by sequencing, and the verification result was correct. At the same time, using rice cDNA as a template, the combination of P1+P4 primers amplifies the wild-type SAPK10 gene (SEQ ID NO: 4), and connects it into PU1301 in the same way to obtain the PU1301-Ubiquitin-SAPK10 overexpression vector.

[0055] 2. Genetic modification. The recipient material for genetic transformation is the japonica rice (Oryza sative L. ssp ja ponica) variety Nipponbare, hereinafter referred to as Nip. PU1301-Ubiquitin-SAPK10 and PU1...

Embodiment 3

[0058] Example 3 Expression detection and sequencing verification of transgenic positive strains

[0059] First select the transgenic positive strain SAPK10 and SAPK10 identified in Example 2 S177A Each of the 2 strains was tested for overexpression of genes, which were recorded as SAPK10(o)-1, SAPK10(o)-2, and SAPK10 S177A (o)-1, SAPK10 S177A (o)-2. Nipponbare and wild-type SAPK10 and SA PK10 were extracted respectively by the Trizol method in Example 1 S177A The total RNA of leaves of each of the 2 lines (the constructed vector is the Ubiqui tin constitutive expression promoter of maize, if it is a transgenic positive plant, then the plant body will overexpress SAPK10 and SAPK10 S177A ), reverse transcribe into cDNA, and measure the expression abundance of transgenic plants by quantitative PCR reaction.

[0060] Table 2. Primers for quantitative detection of SAPK10 transgenic lines

[0061]

[0062] Reagents and qRT-PCR system and procedures:

[0063] Master is Bio-...

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Abstract

The invention discloses a phosphorylated protein kinase SAPK10 mutant and a method of the phosphorylated protein kinase SAPK10 mutant. The mutant is obtained by mutating serine on the 177th site of the SAPK10 to alanine, specifically, a possible phosphorylation site of the SAPK10 protein sequence is modified through genetic engineering; the serine, an amino acid residue, on the177th site is mutated to the alanine; and the modified SAPK10 protein is obtained. The method comprises the step of determining the phosphorylation site of the non-glycolysis type protein kinase SAPK10 of rice saccharose; the phosphorylation site is modified through genetic engineering, a Ubiquitin promoter of corn drives, compared with the transgenic rice whose phosphorylation site is not modified after the transgenosis, the sensitivity to ABA is reduced, the specific performances are on the agronomic characters, the plant height, the grain shape, the maturing rate, the thousand seed weight, the number of productive ear, and the ear length recover to the levels of the wild type, moreover, the ABA also shows the same trend on the influence on the germination capability of the seed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and crop genetic improvement, and specifically relates to a phosphorylated protein kinase SAPK10 mutant and a method thereof. Background technique [0002] SnRK2 is a plant-specific serine / threonine kinase family that is mainly involved in plant stress resistance and ABA-induced signal transduction (Shukla and Mattoo, 2008). Some members of the SnRK2 kinase family have been reported to be induced by ABA (Boudsocq et al., 2004; Kobayashi et al., 2005) and play key roles in plant responses to ABA-mediated plant development and seed dormancy (Johnson et al. al., 2002; Mustilli et al., 2002; Fujii et al., 2007). [0003] Abscisic acid (ABA) is a natural hormone identified in the 1960s. It is a class of 15-carbon acidic sesquiterpenes that widely exist in higher plants. Abscisic acid regulates a variety of plant growth and development and stress resistance processes. When a plant encount...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12
CPCC12N9/1205
Inventor 王以锋张健童晓红
Owner CHINA NAT RICE RES INST