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GhASN-like gene, expression vector and its application in raising cotton output

A plant expression vector and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of unknown gene function, and achieve the effect of simple and easy method, increased cottonseed yield, and improved yield

Inactive Publication Date: 2013-01-16
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Homology analysis shows that the amino acid residues encoded by this gene share more than 75% homology with the amino acid sequences encoded by auxin down-regulation and aluminum-induced genes, but the functions of these genes are currently unknown

Method used

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  • GhASN-like gene, expression vector and its application in raising cotton output
  • GhASN-like gene, expression vector and its application in raising cotton output
  • GhASN-like gene, expression vector and its application in raising cotton output

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] [Example 1] Preparation of target gene

[0039] 1. Extraction of DNA

[0040] Select 0.5-1 g of young cotton leaves, quickly grind them into powder in liquid nitrogen, add 3 mL of CTAB extract (100 mmol / L Tris-HCl (pH8.0), 20 mmol / L EDTA (pH8.0) preheated at 65 °C, 1.5mol / L NaCl, 2% CTAB (W / V), 4% PVP40 (W / V) and 2% mercaptoethanol (V / V), add PVP and mercaptoethanol before use), shake and mix quickly. Bath at 65°C for 30min, then add 1mL of 5mol / L KAc, ice-bath for 20min, extract once with an equal volume of chloroform:isoamyl alcohol (24:1) (10,000r / min, centrifuge at 4°C for 5min), and take the To clean it, add 2 / 3 times the volume of -20°C pre-cooled isopropanol, mix well, and let it stand for about 30 minutes. Rinse once, blow dry, and resuspend in 500 μL TE. Add 1 μL of RNaseA (10 mg / mL) and treat at 37°C for 1 h. Then phenol (pH8.0): chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) were extracted once each (10,000r / min, centrifuged ...

Embodiment 2

[0047] [Example 2] Influence of increasing auxin content on GhASN-like gene expression in vitro and in vivo

[0048] Take the bud on the day of flowering, peel off the petals, sepals, etc., and leave the ovary. The ovary was soaked in 75% alcohol for surface activation treatment, then 0.1% mercuric chloride was added, sterilized for 10-15 minutes, and washed 6 times with sterile distilled water. Strip the ovules and place them in the ovule medium (the normal medium formula for ovule in vitro is: BT medium + 18g / L glucose + 3.6g / L fructose + 0.5μmol / L GA3 + 5μmol / L IAA), and place at 32°C Cultured in the dark in the incubator. The specific plan is as follows:

[0049] (1) Add 2 μmol / L, 5 μmol / L, 10 μmol / L and 25 μmol / L IAA to the culture medium to culture the ovules of ODPA for 5 days.

[0050] (2) ODPA ovules were cultured for 5 days in the medium supplemented with 5 μmol / L IAA, 0.5 μmol / L GA3 and NPA (NPA concentration: 50 μmol / L), an inhibitor of IAA polar transport.

[...

Embodiment 3

[0054] [Example 3] Target gene expression level detection

[0055] After extracting the total RNA from the plant material, a cDNA one-strand cDNA synthesis kit (MBI company) was used to synthesize one-strand cDNA of various RNAs, and all operations were performed according to the instructions of the kit. Take 1 μL of the first-strand product as a template and perform RT-PCR amplification first to detect the template amplification effect. Its 25μL amplification system: including 1×PCR buffer, 0.2mmol / L dNTPs, 1.5mmol / L MgCl2, 0.2μmol / L each of upstream and downstream primers (SEQ ID NO.5 and SEQ ID NO.6), 1U Taq DNA polymerase (Promega). The thermal cycle parameters are: 94°C pre-denaturation for 3 minutes; 94°C, 30sec, 56°C, 30sec, 72°C, 30s, 30 cycles; 72°C extension for 10min. Histone3 gene was used as internal standard. The primer sequences of Histone3 are SEQ ID NO.7 and SEQ ID NO.8 (Zhu YQ et al., 2003, Plant Physiology, 133, 580-88).

[0056] The template with good a...

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Abstract

According to the invention, over-expression vectors are constructed by GhASN-like gene and are integrated into a cotton genome to accomplish the over-expression of the gene in cotton, raise the content of sugar in ovule, and increase plant boll-forming number of a transgenic strain and seeds of each boll, therefore increasing the output of unginned cotton and ginned cotton. By applying the method, the output of unginned cotton and ginned cotton per unit area can be obviously increased, the output of cotton fibers can be raised, and the economic benefit of the cotton production can be raised; and simultaneously, the cottonseed output can be increased so as to lift the added value of the cotton production and raise the comprehensive benefit of the cotton production.

Description

technical field [0001] The invention relates to the use of the cotton GhASN-like gene, and also relates to a plant expression vector for overexpressing the gene and a method for increasing cotton fiber and cottonseed yield through genetic engineering technology. Background technique [0002] Cotton is the most important natural fiber crop in the world, and it is also an important source of protein and oil. It is precisely because cotton fiber and seeds are closely related to human life that it has become the most important economic crop in the world. my country is the world's largest producer and consumer of textiles, and the cotton industry plays a pivotal role in my country's national economy. [0003] Cotton yield and quality traits are quantitative traits controlled by polygenes, and there is a negative correlation between yield and quality traits genetically. Conventional breeding methods have played an important role in the breeding of high-yield and high-quality cot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00
Inventor 李德谋裴炎罗小英侯磊李先碧肖月华罗明白文钦张觅李忠旺梁俊俊
Owner SOUTHWEST UNIV
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