Cotton casein kinase gene and its application

A casein kinase and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of cotton gene cloning and functional verification, complex biological components and genome, etc.

Inactive Publication Date: 2013-02-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The biological components and genome of cotton are very complex, and the gene cloning and functional verification of cotton are also difficult
Most of the existing research

Method used

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  • Cotton casein kinase gene and its application
  • Cotton casein kinase gene and its application
  • Cotton casein kinase gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] GhCKI Gene Isolation and Cloning

[0049] 1. Expression profile analysis

[0050] The applicant's previous work was to perform expression profile analysis on anthers of upland cotton '84021' and 'H05' under high temperature stress (see figure 1 a) Through gene expression clustering analysis, a class of genes with opposite expression trends in the two cotton varieties was isolated, and a sequence highly homologous to CKI in other plants was isolated from this class of genes, and analyzed expression analysis of the sequence (see figure 1 b). Evolutionary analysis and bioinformatics analysis of CKI genes in other species showed that the sequence had the highest homology with Arabidopsis AtCKIL2 (AT1G72710), and it was named GhCKI (see figure 2 ). Total RNA was extracted from the upland cotton lines '84021' and 'H05' before the anthers cracked on the day of flowering under high temperature treatment and normal temperature (the extraction method was based on Zhu et al.,...

Embodiment 2

[0054] Expression Analysis of GhCKI in Different Cotton Tissues

[0055]Using upland cotton YZ1 as a material, the RNA of the following seven different tissues was extracted, and the expression level of GhCKI gene was detected by Rea-time PCR and Northern blot. The seven selected tissues are: 1. petal; 2.0DPA fiber; 3.5DPA fiber; 4. ovule; 5. anther; 6. leaf; 7. stigma. Cotton total RNA extraction method according to the above-mentioned literature published by Zhu et al. (An improved simple protocol for isolation of high quality RNA from Gossypium spp. suitable for cDNA library construction. Acta Agronomica Sinica. Treat with DNaseI (purchased from Promega), and the RNA integrity is detected by 1.2% (w / v) agarose gel (EtBr) electrophoresis (5V / cm). The determination of nucleic acid concentration was carried out on a Beckman DU800spectrophotometer. The RNA 260 / 280 ratio was between 1.9 and 2.1, and the RNA with a 260 / 230 ratio greater than 2.0 was used for the next analysis. ...

Embodiment 3

[0057] Construction and Transformation of Overexpression Vector and RNAi Inhibition Expression Vector

[0058] In order to verify the function of the GhCKI gene, the applicant constructed an overexpression and RNAi suppression expression vector to transform cotton, and also transformed Arabidopsis thaliana with the overexpression vector.

[0059] According to the GhCKI ORF obtained in Example 1, the overexpression primers were designed, and the enzyme cleavage sites and protective bases of XbaI and SacI were respectively added to the two ends of the primers, and the primers were respectively named as primers GhCKIoe-F and GhCKIoe-R, and then The cDNA of the GhCKI gene was used as a template for PCR amplification, and the obtained PCR product was completely digested with restriction endonucleases XbaI and SacI (NEB Company), and the digested product was electrophoresed on a 0.8% agarose gel (80V, 40 minutes), A DNA fragment of about 1.4kb was excavated and recovered. Take 100n...

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Abstract

The invention discloses a cotton casein kinase gene and its application, and relates to a separation cloning, a function verification and an application of a gene GhCKI used for controlling anther growth and identified in a cotton anther expression spectrum. There is a 72% homology between the protein coded by the GhCKI gene and the protein coded by an Arabidopis thaliana ATCKL2 gene, and the GhCKI gene preferentially expresses in the anther, has an anther growth controlling function, can be applied to the sterile line cultivation of a plurality of plants through genetic transformation, and simultaneously can be used as a marker gene of the plant anther fertility stability under high temperature stress.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. It specifically relates to a new cotton casein kinase gene, adopts the method of reverse genetics, analyzes the anther expression profile under high temperature stress of cotton, and isolates and clones the GhCKI gene as a gene controlling the development of cotton anthers. The present invention also relates to a vector containing the gene or its homologous gene and the application of the gene or its functional analog to regulate anther development of plants in agricultural production. Background technique [0002] Temperature is an important environmental factor affecting the distribution and growth of plants. However, with the rapid development of industrialization, a large amount of greenhouse gas emissions, the aggravation of global warming, and the increase in the frequency of high-temperature weather, plant growth is facing severe challenges from high-temperature stress. . General...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/84A01H5/00
Inventor 张献龙朱龙付闵玲涂礼莉
Owner HUAZHONG AGRI UNIV
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