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Reproductive organ and glandular hair tissue specific promoter GhS and application thereof

A promoter and biological technology, applied in the application field of plant genetic improvement, can solve problems such as less research and more in-depth research

Active Publication Date: 2018-07-13
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although scientists have cloned the above-mentioned promoters related to seed-specific expression in different plants, so far, there are few studies on the promoters specific to reproductive organs and cotton fiber tissues, and further research is needed

Method used

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  • Reproductive organ and glandular hair tissue specific promoter GhS and application thereof
  • Reproductive organ and glandular hair tissue specific promoter GhS and application thereof
  • Reproductive organ and glandular hair tissue specific promoter GhS and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning of seed coat and fibrous tissue-specific promoter GhS

[0043] (1) DNA Extraction of Upland Cotton (Gossypium hirsutum)

[0044] 1) Take fresh cotton leaves and put them in a centrifuge tube with steel balls, freeze them quickly in liquid nitrogen, and process them with a sample rapid grinder at 55Hz for 100 seconds until the leaves are completely broken.

[0045] 2) Add 600 μL of CTAB extract (containing 2% β-mercaptoethanol), mix evenly by inversion, and bathe in a water bath at 65° C. for 30-60 minutes.

[0046] 3) Add an equal volume of chloroform:isoamyl alcohol (24:1), gently invert and mix, let stand for 10 minutes; centrifuge at 12000rpm for 10 minutes at 4°C;

[0047] 4) Transfer the supernatant to a new centrifuge tube, add 2 times the volume of absolute ethanol, mix it upside down, and place it at -20°C for more than 30 minutes;

[0048] 5) Centrifuge at 12,000 rpm for 10 minutes, discard the supernatant; add 700 μL of 75% ethanol, and r...

Embodiment 2

[0058] Embodiment 2: GhS promoter plant expression vector construction

[0059] The strains sequenced correctly in Example 1 were selected to extract plasmids for the construction of plant expression vectors. The above-mentioned pMD18-T recombinant vector plasmid linked to the GhS promoter and the empty pCAMBIA1305 plasmid were subjected to BamI and NcoI double digestion respectively. Digest at 37°C for 15-30 minutes, and perform agarose gel electrophoresis detection on the digested products, recover the GhS promoter fragment digested by the recombinant plasmid and the large vector fragment digested by pCAMBIA1305, and connect them. The connection system is as follows:

[0060]

[0061] Ligate overnight at 16°C, then transform into Escherichia coli DH5α by heat shock method, pick clones for PCR detection, and send positive clones for sequencing verification;

[0062] The completed GhS-pCAMBIA1305 promoter expression vector is shown in figure 1 . In this figure, the GFP...

Embodiment 3

[0063] Example 3: Tissue expression analysis of GUS gene driven by GhS promoter in Arabidopsis

[0064] (1) Genetic transformation of GhS-pCAMBIA1305 vector

[0065] 1) Transformation of Agrobacterium with GhS-pCAMBIA1305 vector

[0066] The strains sequenced correctly in Example 2 were selected, and the plasmids were extracted for subsequent Agrobacterium transformation. The method is similar to transforming E. coli. Take 2 μL of the recombinant plasmid and add it to 100 μL of Agrobacterium GV3101 competent cells, mix gently and let stand on ice for 30 minutes. Immediately after liquid nitrogen treatment for 3 minutes, heat shock at 37°C for 10 minutes, followed by ice bathing for 1 to 3 minutes. Add 800 μL LB non-resistance liquid medium, and incubate on a shaker at 180 rpm at 28°C for 3 to 5 hours. After centrifuging at 4000rpm for 1 minute, discard most of the supernatant, suspend the bacteria and spread evenly on a solid LB kanamycin-resistant plate, incubate at 28°...

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Abstract

The invention belongs to the field of plant gene engineering and discloses a reproductive organ and glandular hair tissue specific promoter GhS and the application of the promoter. The promoter is specifically express in arabidopsis thaliana anthers and stigmatoid tissues, petals, sepal glandular hair, leaf glandular hair, seed funiculi and epidermal hair of part of seeds. A nucleotide sequence ofthe promoter GhS is shown as SEQ ID NO. (sequence identifier number) 1. The promoter GhS has the following characteristics: A, an expression situation in an arabidopsis thaliana tissue is very clear;the expression of the promoter on plants such as cotton is not reported; B, the sequence of the promoter is from a cotton ovule; the specific expression of the promoter in the arabidopsis thaliana glandular hair implies that the promoter can specifically regulate the expression of a target gene in cotton fibers; and C, the promoter is novel in structure and has no homology with other promoters reported on a nucleic acid level; and homology sites do not contain the existing patent protected sequences and mutant sites.

Description

technical field [0001] The present invention relates to a reproductive organ and glandular hair tissue-specific promoter GhS and its application, specifically a promoter proGhS (GhS promoter for short) that can be specifically expressed in plant reproductive organs and glandular hair tissue, and the promoter can be used in plants. applications in genetic improvement. Background technique [0002] Plant development refers to the process of changing the structure and function of plants from simple to complex. In this complementary process, it is always controlled by a series of internal and external factors. At the molecular level, the growth and life cycle of plants is a comprehensive phenomenon of the orderly expression and synergy of different genes in time and space based on various metabolic and physiological processes in plants. Gene switching, expression patterns, and expression abundance are all regulated precisely throughout the life cycle. The regulation of gene ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/10C12N15/63C12N1/21C12R1/19C12R1/01
CPCC07K14/415C12N15/823
Inventor 左开井孙文杰高正银陈云
Owner SHANGHAI JIAO TONG UNIV
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