Screening method for artemisia japonica plasma membrane Na+/H+ reverse transport protein AjSOS1 salt tolerance key amino acid site

A site, pag426gpd-sos1s technology, applied in the direction of microorganism-based methods, DNA preparation, recombinant DNA technology, etc., can solve the problems of lack of key amino acid screening of genes and weak research

Inactive Publication Date: 2016-06-29
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0025] In view of the relatively weak research on the molecular mechanism of salt tolerance of chrysanthemum and its close relatives, and the lack of technical problems in the prior art about the screening of key amino acids in genes, the purpose of the present invention is to provide a recombinant expression of SOS1s gene carrier

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  • Screening method for artemisia japonica plasma membrane Na+/H+ reverse transport protein AjSOS1 salt tolerance key amino acid site
  • Screening method for artemisia japonica plasma membrane Na+/H+ reverse transport protein AjSOS1 salt tolerance key amino acid site
  • Screening method for artemisia japonica plasma membrane Na+/H+ reverse transport protein AjSOS1 salt tolerance key amino acid site

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Embodiment 1

[0090] (1) Cloning of SOS1s gene of chrysanthemum and its three relative species

[0091] Trizol reagent (TaKaRa) was used according to the instructions to treat the highly salt-tolerant Hibiscus chrysanthemum and Artemisia annua under the treatment of 200mM NaCl; Yong. Salt tolerance screening of 32 chrysanthemum relatives. Chinese Agricultural Sciences (2010).】The total RNA was extracted from the roots of 4 kinds of plants, and Oligod(T) 18 As a reverse transcription primer, perform reverse transcription according to the instructions of the M-MLV reverse transcription kit (TaKaRa) to obtain the first-strand cDNA template, and use Premier 5.0 software to design specific primers according to the sequence information of the gene in the chrysanthemum library:

[0092] Upstream primer F1: the sequence is shown in SEQ ID NO.5,

[0093] Downstream primer R1: the sequence is shown in SEQ ID NO.6;

[0094] Upstream primer F2: the sequence is shown in SEQ ID NO.7,

[0095] Downstre...

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Abstract

The invention belongs to the technical field of genetic engineering and biology, and discloses a screening method for an artemisia japonica plasma membrane Na+/H+ reverse transport protein AjSOS1 salt tolerance key amino acid site. Transformation of SOS1s gene recombinant vectors, transformed cells and yeast mutants and salt tolerance analysis of the transformed cells are provided. The screening method for the AjSOS1 salt tolerance key amino acid site comprises the steps of 1, cloning of an SOS1s gene; 2, construction of yeast expression vector pAG426GPD-SOS1s; 3, salt tolerance analysis of four SOS1s yeast transformed cells; 4, screening of the AjSOS1 salt tolerance key amino acid site. A series of AjSOS1muts are generated through site-specific mutagenesis, and anaplerosis is performed on yeast mutants ANT3, the salt tolerance key amino acid site in an AjSOS1 sequence is found, a novel and practical method is provided for research of chrysanthemum and affinis species plant salt tolerance molecular mechanism, and the breeding process of the chrysanthemum biological technology is effectively promoted.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biotechnology, and relates to a method for screening key amino acid sites of target genes through amino acid site-directed mutagenesis and complementation of yeast mutants, specifically involving AjSOS1, CrcSOS1, CcSOS1 and CmSOS1 genes (collectively referred to as SOS1s) recombinant vector, transformation of yeast mutants, analysis of salt tolerance of 4 SOS1s-transformed yeast cells, generation of AjSOS1 site-directed mutations, and AjSOS1muts yeast-transformed cells Na + Comparative analysis of differences in transport capacity. Background technique [0002] Soil salinization is one of the important abiotic stresses affecting crop yield and quality in agricultural production. Chrysanthemum is one of my country's top ten traditional famous flowers and one of the world's four major cut flowers. It has a wide range of uses and cultivation areas, and has high ornamental and econom...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/81C12N15/01C12N15/66C12N1/19C12Q1/68C12Q1/02C12R1/84
CPCC07K14/415C12N15/102C12N15/66C12N15/81C12N2800/102C12Q1/02C12Q1/6858C12Q2531/113C12Q2565/125C12Q2535/125
Inventor 蒋甲福高姣姣刘晨
Owner NANJING AGRICULTURAL UNIVERSITY
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