Cotton fiber predominantly-expressed gene, expression vector and applications thereof, and preparation method of transgenic cotton containing gene

A cotton fiber and gene expression technology, applied in the field of plant genetic engineering, can solve problems such as molecular design that cannot meet the cotton fiber yield and quality

Inactive Publication Date: 2015-05-13
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, the obtained fiber-specific or predominantly expressed genes are still too few to meet the needs of molecular design for improving cotton fiber yield and quality

Method used

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  • Cotton fiber predominantly-expressed gene, expression vector and applications thereof, and preparation method of transgenic cotton containing gene
  • Cotton fiber predominantly-expressed gene, expression vector and applications thereof, and preparation method of transgenic cotton containing gene
  • Cotton fiber predominantly-expressed gene, expression vector and applications thereof, and preparation method of transgenic cotton containing gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Cloning and sequence analysis of the GhMSMO2-2 gene:

[0073] 1. Extraction of Cotton RNA

[0074] Select about 3g of fresh cotton material, quickly grind it into fine powder in liquid nitrogen, put it into a 50mL centrifuge tube, add 15mL of 65°C preheated RNA extraction solution (the RNA extraction solution contains 2% CTAB cetyl trimethyl Ammonium bromide (W / V), 2% PVP polyvinylpyrrolidone (W / V), 100mmol / L Tris-HCl (pH 8.0), 0.5g / L Spermidine spermidine solution, 2.0mol / LNaCl, 2% mercapto Ethanol (V / V, added before use)), in a water bath at 65°C for 3 to 10 minutes, and mixed 2 to 3 times during this period; then extracted twice with a mixed solution of chloroform:isoamyl alcohol (24:1v / v), And after centrifuging at 10,000r / min at room temperature for 5min, take the supernatant, add 1 / 4 volume of the supernatant to 10mol / L LiCl solution, place it at 4°C for 6h, add chloroform:isoamyl alcohol (24:1v / v ) each extracted once, centrifuged at 10,000r / min at r...

Embodiment 2

[0095] Example 2: Expression analysis of GhMSMO2-2 gene:

[0096] 1. Expression analysis of GhMSMO2-2 gene in cotton plant and fiber development

[0097] Total RNA was extracted from various tissues and organs of upland cotton (Gossypium hirsutum L.), and one strand of cDNA was synthesized. Using the synthesized cDNA strand as a template, real-time quantitative PCR kit (Bio-Rad) was used for expression analysis. The 5' end primer of GhMSMO2-2 gene is P3 (SEQ ID NO.6), and the 3' end primer is P4 (SEQ ID NO.7). Include 10 μL MIX buffer (including PCR buffer, DNA polymerase, dNTPs, provided by real-time quantitative RT-PCR kit, Bio-Rad) in a 20 μL reaction system, 1 μL each of 5’-end and 3’-end expression primers (5μmol / L). The cycle parameters are 94°C pre-denaturation for 3 minutes; 94°C, 30sec, 55°C, 30sec, 72°C, 30sec, and the preset cycle number is 40. The cotton GhHISTONE3 gene (GenBank accession number: AF024716) was used as an internal standard, the 5'-primer of the ...

Embodiment 3

[0104] Example 3 Construction of overexpression and antisense suppression GhMSMO2-2 gene plant expression vector:

[0105] Figure 9 It is a schematic diagram of the general model structure of the expression vector of the plant containing the GhMSMO2-2 gene, and the corresponding elements are specified in the following specific implementations;

[0106] 1. Construction of overexpression vector and antisense suppression GhMSMO2-2 gene plant expression vector

[0107] The pMD19-GhMSMO2-2 vector was constructed when the GhMSMO2-2 gene was cloned, and the GhMSMO2-2 fragment on it was sequenced. The plant expression vector is a modified pCambia vector (Guangzhou Jisai Biotechnology Co., Ltd.), the HPT II gene of the vector is cleaved with XhoI and replaced with the NPT II gene (the designed primers were amplified from the pBI121 vector, and the primers were designed at both ends with XhoI site), restriction enzyme digestion and sequencing results verified the direction of the NPT ...

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Abstract

The invention discloses a cotton fiber predominantly-expressed gene GhMSMO2-2, which has one of the following nucleotide sequences: a cDNA sequence shown in SEQ ID NO.1, a gDNA sequence shown in SEQ ID NO.2, and a nucleotide sequence which has over 85% homology with the DNA sequences above and encodes functional proteins same as those encoded by the DNA sequences above, or a nucleotide sequence hybridized with the nucleotide sequences above under high-preciseness conditions; a protein encoded by the gene has an amino acid sequence shown in SEQ ID NO.3; the invention also provides a plant expression vector of the gene which at least contains a promoter sequence and the nucleotide sequences of the gene; the invention further provides a preparation method of a transgenic cotton containing the gene, which is implemented by transforming a host by using the vector, so that a transformant is obtained; and transforming a cotton by using the transformant, so that the transgenic cotton is obtained. The GhMSMO2-2 gene can be applied to the promotion of the plant growth of the transgenic cotton, the increasing of fiber length, and the improvement of fiber quality and yield.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a cotton fiber dominant expression gene, a coding protein containing the gene and a plant expression vector, and also relates to the application of the above gene and a preparation method for transgenic cotton containing the above gene. Background technique [0002] Cotton is one of the world's major fiber crops. Cotton fiber cells are single cells formed by differentiated protrusions and polar elongation of epidermal cells in the outer integument of cotton ovules. Its growth and development process is divided into four distinct and overlapping periods: fibroblast initiation period, fibroblast elongation period, secondary wall thickening period and dehydration maturation period. Each period has its own characteristics, but adjacent developmental periods overlap. Each stage of fiber development has an effect on the yield and / or quality of fibers. The initiatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/84A01H5/00
Inventor 罗明隗廷李芳翟云兰曾志锋唐英才裴炎肖月华侯磊
Owner SOUTHWEST UNIV
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