Cotton cell wall extensin gene gbeexpatr and its application

A technology of extensin and cell wall, which is applied in cotton cell wall extensin gene GbEXPATR and its application field, can solve the problems of unclear biological functions, and achieve the effect of promoting elongation and high accuracy

Active Publication Date: 2016-02-17
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They may have evolved from β-expansin, which is 35-45% similar to β-expansin, but its biological function is still unclear

Method used

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  • Cotton cell wall extensin gene gbeexpatr and its application
  • Cotton cell wall extensin gene gbeexpatr and its application
  • Cotton cell wall extensin gene gbeexpatr and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: GbEXPATR gene isolation clone and its expression characteristics in different tissues of cotton

[0034] In the preliminary work of the applicant's State Key Laboratory of Crop Genetic Improvement, the cDNA library of sea island cotton fiber development in different periods (Tu, L.L.; et al. Genes expression analyzes of sea-island cotton (Gossypium barbadense L.) during fiber development. .) isolated a cDNA that is highly homologous to the upland cotton cell wall expansin α-expansin gene (gene accession number: AF043284) but lacks the second domain (see SEQ ID NO: 1). Bioinformatics analysis showed that the cDNA Contains a complete ORF (see SEQ ID NO: 3), which can be translated into an incomplete α-expansin protein through bioinformatics prediction (see SEQ ID NO: 2 for the amino acid sequence of the encoded protein), we named this fragment is the GbEXPATR gene. The gene is highly expressed at various stages of fiber elongation, and 10DPA fiber total RNA wa...

Embodiment 2

[0035] Embodiment 2: Construction and transformation of overexpression vector

[0036] In order to verify the function of the GbEXPATR gene in cotton, the applicant constructed an overexpression vector to transform cotton.

[0037] According to the ORF region of GbEXPATRcDNA obtained in Example 1 (see the region corresponding to 52-306 bases in SEQ ID NO: 1), the overexpression primer pair was designed (the DNA sequence of the primer pair is described in this paragraph), and we used two primer pairs NcoI and BstEII enzyme cutting sites and protective bases were added to the ends of the primers respectively, and the primers were named as primers GbEXPATRPoef and GbEXPATRPoer, and then PCR amplification was performed using the cDNA of the GbEXPATR gene as a template, and the obtained PCR products were ligated by enzyme digestion. The method was ligated into pCAMBIA2301 digested with the same enzyme m on the vector (pCAMBIA2301 m The precursor of the vector is pCAMBIA2301 donat...

Embodiment 3

[0070] Example 3: Correlation Analysis of GbEXPATR Transgenic Lines

[0071] 1. Molecular identification of GbEXPATR transgenic lines

[0072] Three transgenic families were obtained through the overexpression of GbEXPATR gene, which were named as OE2, OE3 and OE4. Genomic DNA was extracted from the obtained transgenic families (according to conventional methods), and analyzed by Southern blot to identify the transgenic effect and copy number. Genomic DNA extraction methods and Southern experiments of transgenic families refer to the methods reported by J. Sambrook et al., translated by Huang Peitang et al., Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002 edition. see results Figure 4 shown.

[0073] 2. Expression analysis of GbEXPATR transgenic lines

[0074] Extract the total RNA of the transgenic line by single plant (J. Sambrook et al., Huang Peitang et al. translation, Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002 edi...

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Abstract

The invention belongs to the technical field of plant genetic engineering and particularly relates to cell wall extensin gene GbEXPATR separated and cloned from cotton and application. The cell wall extensin gene GbEXPATR which is obtained through sea island cotton fiber specific expression in a cDNA library at different sea island cotton fiber development stages and lack of a second structural domain, and a nucleotide sequence is represented by SEQ ID NO:1, wherein 52-306 represents a basic group region which is a coding region and exceptionally and efficiently expressed in a sea island cotton fiber elongation period, a conversion period and a secondary wall synthesis initial stage. An obtained overall-length ORF is established on a plant excessive expression vector Pcambia2301M, sea island cotton is converted into upland cotton by using an agrobacterium-mediated transformation genetic transformation method, the quality of transgenic offspring fiber is analyzed, and the cell wall extensin gene GbEXPATR can effectively promote fiber elongation is proved. A Micron-aire reduced. The fiber quality is improved from the grade C2 to the grade B2. The breakage ratio and the strength are improved. The cotton fiber quality can be effectively improved by utilizing the cell wall extensin gene GbEXPATR.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to a cotton cell wall extensin gene GbEXPATR and its application. Using the method of reverse genetics, the gene GbEXPATR lacks the second domain of cell wall expansin (α-expansin) through the analysis of the cDNA library of sea island cotton fiber at different developmental stages. The GbEXPATR gene isolated and cloned in the present invention is predominantly expressed in the fiber elongation period, conversion period and the initial stage of secondary wall synthesis of sea-island cotton. Overexpression of this gene in cotton can effectively promote the elongation of fibers, reduce the micronaire value, increase the fiber grade from C2 to B2, and increase the specific strength at break. The invention can effectively improve the cotton fiber quality. Background technique [0002] Cotton is one of the most important economic crops in the world. It prov...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82
Inventor 张献龙李阳涂礼莉袁道军
Owner HUAZHONG AGRI UNIV
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