Application of cotton fat transporter gene GhFIL in improvement of cotton fiber quality

A technology for transporting proteins and cotton fibers, applied in the field of biotechnology applications, can solve problems such as unclear sphingolipids, and achieve the effect of improving the quality of cotton fibers

Active Publication Date: 2022-05-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, how sphingolipids functi

Method used

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  • Application of cotton fat transporter gene GhFIL in improvement of cotton fiber quality
  • Application of cotton fat transporter gene GhFIL in improvement of cotton fiber quality
  • Application of cotton fat transporter gene GhFIL in improvement of cotton fiber quality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 The full-length sequence of the cotton lipid transporter GhFIL was obtained:

[0065] By constructing the 7124-3-6DPA ovule / fiber cDNA library (GB1) and the 6-24DPA fiber cDNA library (GB2), the differential expression analysis of EST showed that there were 2260 unigenes in the two libraries. Based on bioinformatics analysis and gene structure and function research, nine genes encoding 120 amino acid sequences and encoding lipid transfer protein were obtained, which were named LTP1-LTP9 respectively. Among them, LTP4 is obviously dominantly expressed in the stage of fiber development, so it is named as FIL. Therefore, according to the existing sequence, PCR amplification primers were designed to amplify the full-length ORF of the gene (GhFIL) from upland cotton TM-1. The primers used for amplification are listed in Table 1.

[0066] Table 1: Primers used for amplification

[0067]

Embodiment 2

[0068] Example 2 Quantitative RT-PCR Analysis of Cotton Fat Transporter Gene GhFIL

[0069] Design specific primers for GhFIL:

[0070] F:5'-TTACTTGACAGGGAATGGTGC-3'

[0071] R: 5'-GTCCGCTTGCAATACCATAG-3'

[0072] In different tissues and organs, quantitative qRT-PCR detection was carried out. The result shows that this gene is expressed very low in root, stem, and leaf, and it is differentially expressed in different stages of fiber development, wherein the expression level gradually increases in the 8-15 days of the rapid fiber elongation period, and the subsequent expression level gradually decreases (such as figure 1 and figure 2 shown).

Embodiment 3

[0073] Example 3 Verification of transgenic function of cotton lipid transporter gene GhFIL

[0074] pCAMBIA2301 is a traditional plant binary expression vector, and its promoter is the promoter of cotton fiber predominantly expressed gene RDL. Based on this, a sense-antisense binary vector of RDL promoter was constructed, and the insertion position of the sense-antisense fragment was between RDL-P (promoter) and Nos-T (terminator).

[0075] Construct the sense and antisense plant expression vector with the full-length fragment of the sequence of the gene SEQ NO ID.1 of the present invention. After the construction is completed, transform cotton through the Agrobacterium-mediated method for functional verification, and obtain the sense and antisense transgenic plants of the RDL promoter. The above two vectors All transgenic plants have been identified by PCR (such as image 3 shown). Expression analysis found that in the sense vector transgenic plants, the expression of this...

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Abstract

The invention discloses an application of a cotton fat transporter gene GhFIL in improvement of cotton fiber quality. The lipid transporter GhFIL is an important gene in the cotton fiber development process and plays an important role in extension of fiber cells. It is reported for the first time that the gene participates in sphingolipid transport in the cotton fiber development process, and an auxin response pathway is activated. The gene is used as a target gene to construct an antisense plant expression vector for performing transgenic cotton function verification, and the result shows that the content of fatty acid and auxin in fibers is increased, the length and strength of the fibers are remarkably increased, and the micronaire value is remarkably reduced by overexpressing the gene; by inhibiting the gene expression, the content of fatty acid and auxin in the fiber is reduced, the length and strength of the fiber are remarkably reduced, and the micronaire value is remarkably increased. Therefore, overexpression of the gene in cotton can significantly improve the cotton fiber quality.

Description

technical field [0001] The invention belongs to the application field of biotechnology, and relates to the application of the cotton fat transporter gene GhFIL in improving the quality of cotton fibers. The cotton lipid transporter gene GhFIL ORF has a full length of 363bp, encodes a lipid transporter containing 120 amino acids, has 8 conserved cysteine ​​residues, and has a typical signal peptide structure at the N-terminus, with an isoelectric point of 8.8. Through qRT-PCR analysis, it was found that the gene was predominantly expressed during fiber elongation. The gene was expressed in upland cotton (Gossypium hirsutum) Li's ultra-short fiber mutant Li1 (G.hirsutum acc.Li1) and Li1 wild type with normal fiber development, upland cotton genetic standard line TM-1 (G. 1) There are significant differential expressions in the fiber elongation stage, and the full-length ORF sequence of the gene is obtained in TM-1 by PCR technology. Using biotechnology to clarify the gene's in...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/08A01H6/60
CPCC12N15/8247C12N15/8261C07K14/415Y02A40/146
Inventor 郭旺珍段宇佳尚小光
Owner NANJING AGRICULTURAL UNIVERSITY
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