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

A plant expression vector, cotton technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as unknown gene function, achieve the effect of simple and easy method, increase yield, and promote ovule development

Inactive Publication Date: 2011-10-19
SOUTHWEST UNIVERSITY
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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. DNA extraction

[0040] Select 0.5-1 g of young cotton leaves, quickly grind 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, PVP and mercaptoethanol were added before use), and the mixture was quickly shaken and mixed. Water 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), take the Add 2 / 3 times the volume of -20 ℃ pre-cooled isopropanol, mix well, let stand for about 30 minutes, pick out the flocculent precipitate with a glass rod, rinse several times with 75% ethanol, and then use anhydrous ethanol Rinse once, blow dry, and resuspend in 500 μL TE. 1 μL of RNaseA (10 mg / mL) was added and treated at 37°C for 1 h. Then use phenol ...

Embodiment 2

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

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

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

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

[005...

Embodiment 3

[0054] [Example 3] Detection of target gene expression level

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

[0056] The templat...

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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 present invention relates to the use of cotton GhASN-like gene, and also relates to a plant expression vector overexpressing the gene and a method for improving cotton fiber and cotton seed yield through genetic engineering technology. Background technique [0002] Cotton is the most important natural fiber crop in the world and an important source of protein and oil. It is precisely because cotton fibers and seeds are closely related to human life that it has become the most important economic crop in the world. my country is the largest textile producer and consumer in the world, and the cotton industry plays an important role in our national economy. [0003] The yield and quality traits of cotton 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 cotton with high yield and high quality....

Claims

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Application Information

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Patent Type & Authority Applications(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 UNIVERSITY
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