Fusion gene for regulating and controlling color of cotton fibers and expression vector and application of fusion gene

A technology that integrates genes and cotton fibers, and is applied in the fields of application, fusion peptides, and genetic engineering, and can solve problems that cannot meet the needs of consumers and the diversity of colors in the textile industry.

Active Publication Date: 2020-10-27
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the colored cotton materials used in textiles are only green and brown, w

Method used

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  • Fusion gene for regulating and controlling color of cotton fibers and expression vector and application of fusion gene
  • Fusion gene for regulating and controlling color of cotton fibers and expression vector and application of fusion gene
  • Fusion gene for regulating and controlling color of cotton fibers and expression vector and application of fusion gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Acquisition of maize leaf color gene Lc gene, small fragment peptide 2A peptide gene, cotton anthocyanin synthesis regulation gene GhPAP1D

[0034] The small fragment peptide 2A peptide gene and the Lc gene were synthesized by Huada optimized fusion, and the synthesized fragment was used as a template to design primers (Table 1, Lc-2A-U and Lc-2A-D). The 5' of the primer Lc-2A-U A Bam HI restriction site was added to the end, and a 10bp homology arm of the GhPAP1D gene was added to the 5' end of Lc-2A-D. The amplification system is preferably a 10 μl system, including: 2×PrimeSTAR MAX Premix 5 μl, primers Lc-2A-U and Lc-2A-D (5 μmol / L) each 1 μl, template DNA about 60ng, add ddH 2 0 to 10 μl. The amplification program was as follows: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 60 s, 35 cycles; and finally extension at 72°C for 10 min.

[0035] Using the CDS sequence of Gohir.D7G082100 as a r...

Embodiment 2

[0040] Construction of a Plant Expression Vector Specifically Expressing Lc and GhPAP1D Fusion Gene During Fiber Secondary Wall Synthesis

[0041] The process of constructing the coding sequences of Lc-2A and GhPAP1D genes into the plant expression vector pLGN is shown in figure 2 . pLGN is a binary plant expression vector transformed from the traditional plant expression vector pBI121. Its T-DNA segment (region between RB and LB, figure 2 ) was replaced by a fusion gene expression cassette of reporter gene GUS and marker gene NPTII controlled by a constitutive 2×35S promoter (2×35S-P). Use primers pFbl2A-F and pFbl2A-R (see Table 1 for the sequence) to amplify and clone the promoter Fbl2A from the genome of the cotton line Jimian No. 14, add a Hind III restriction site to the 5' end of the promoter, and start A BamH Ⅰ restriction site was added to the 3' end of the sub. The amplification system of the present invention is preferably a 10 μl system, including: 5 μl of 2×...

Embodiment 3

[0046] genetic transformation of cotton

[0047] The cotton genetic transformation of the above expression vector was carried out by the method mediated by Agrobacterium tumefaciens, and the medium formula used is shown in Table 2. The specific method is as follows: shell the full cotton seeds of wild type upland cotton YZ-1, put a small amount (about 20 to 40 seeds) of shelled seeds in a sterilized 100mL triangular flask, and first wash them with 75% alcohol Pre-wash the seeds for 1 min, pour off the alcohol and add 0.1% HgCl 2 Sterilize for about 12 minutes (constantly shake the Erlenmeyer flask to sterilize), gently pour out the mercuric chloride, add sterile water to fully rinse the seeds, and rinse for about 10 times. Place the sterilized seeds on the germination medium, and after the radicle grows about 1 cm (about 36-48 hours), gently insert the radicle into the germination medium, and cultivate the hypocotyl in the dark at about 30°C to about 7 cm (about 7 days). Ab...

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Abstract

The invention provides a fusion gene for regulating and controlling color of cotton fibers and an expression vector and application of the fusion gene, and relates to the technical field of transgenicbreeding of plants. A 2A peptide capable of being self-cracked into small-fragment peptides is utilized, Lc and GhPAP1 genes are synthesized into the fusion gene, a fiber secondary wall specific promoter pFbl2A is adopted to start excessive expression of the fusion gene in the cotton fibers, and the anthocyanin content of the obtained transgenic cotton fibers is remarkably increased compared withthat of white fibers; the total anthocyanin content in the transgenic fiber is increased by 4093 times compared with that in the white fiber, so that the transgenic cotton generates purple red fibers, and the color range of the mature fibers is obviously expanded.

Description

technical field [0001] The invention belongs to the technical field of plant transgenic breeding, and in particular relates to a fusion gene for regulating the coloration of cotton fibers, an expression carrier and application thereof. Background technique [0002] Natural colored cotton is a cotton material that can synthesize and accumulate natural pigments during fiber development, so that mature fibers have natural colors. Compared with conventional white cotton and chemical fibers, colored cotton does not require bleaching and printing and dyeing in the textile processing process due to its natural color, avoiding the pollution of textiles and the harm to human body by toxic and harmful residues such as dyes, organic solvents, and heavy metals. , can significantly improve the green production level of textiles. At present, the colored cotton materials used in weaving are only green and brown, which cannot meet the needs of consumers and the textile industry for color d...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/84A01H5/10A01H6/60
CPCC07K14/415C12N15/8205C12N15/8234C12N15/825C07K2319/00
Inventor 冉玲芳肖月华李耀华曾健晏王毅莫童梁爱敏罗明
Owner SOUTHWEST UNIVERSITY
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