Recombinant vector and expression method of tea tree heat shock protein CssHSP-6 gene

A heat shock protein and recombinant vector technology, applied in the biological field, can solve the problems of cumbersome steps and low success rate, and achieve the effect of improving disease resistance

Inactive Publication Date: 2019-08-13
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has cumbersome steps and a low success rate

Method used

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  • Recombinant vector and expression method of tea tree heat shock protein CssHSP-6 gene
  • Recombinant vector and expression method of tea tree heat shock protein CssHSP-6 gene
  • Recombinant vector and expression method of tea tree heat shock protein CssHSP-6 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction and Identification of Tea Tree Heat Shock Protein CssHSP-6 Gene Recombination Vector

[0029] 1. Extract RNA from tea tree leaves and reverse transcribe cDNA

[0030] Tea tree leaf materials were taken, and total RNA at the seedling stage was extracted with an RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and cDNA was reverse-transcribed with a reverse transcription kit (Promega).

[0031] 2. Using cDNA as a template to amplify the CssHSP-6 gene;

[0032] Primers were designed to amplify the CssHSP-6 gene using cDNA as a template. The nucleotide sequence of the CssHSP-6 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the CssHSP-6 gene is shown in SEQ ID NO. 2.

[0033] Primers are as follows:

[0034] Upstream primer: CssHSP-6-F: GC GAATTC The underline of ATGGCGATGATTCCAAG is the EcoRI restriction site;

[0035] Downstream primer: CssHSP-6-R: CG CTCGAG TCAACCAGATATCTCAATAGACTT...

Embodiment 2

[0042] Example 2: Induced expression of CssHSP-6 protein

[0043] 1. Obtain the recombinant prokaryotic expression strain of CssHSP-6

[0044] The single clone successfully sequenced in Example 1 was selected and inoculated into 50ug / mL kanamycin liquid medium, cultured overnight at 37°C and 200rpm, and the pET-28a - The CssHSP-6 recombinant expression vector was extracted, and the recombinant expression vector was transformed into Escherichia coli BL21(DE3), Rosetta(DE3), BL21(DE3)pLysS strains, and the expression of CssHSP-6 protein was detected.

[0045] 2. Cultivate the activated strain overnight

[0046] The above-mentioned recombinant prokaryotic expression strains were activated by culturing overnight. For example, transfer BL21(DE3) strains to 50ug / mL kanamycin liquid medium, transfer Rosetta(DE3), BL21(DE3)pLysS strains to 50ug / mL kanamycin+50ug / mL chlorine The activated strain was cultured overnight at 37°C and 200 rpm in a mycin liquid medium.

[0047] 3. Induced ...

Embodiment 3

[0055] Example 3: Verification of CssHSP-6 Protein Inducible Expression Conditions

[0056] 1. Obtain the recombinant expression strain of CssHSP-6

[0057] The single clone successfully sequenced in Example 1 was selected and inoculated into 50ug / mL kanamycin liquid medium, cultured overnight at 37°C and 200rpm, and the pET-28a - The CssHSP-6 recombinant expression vector was extracted, and the recombinant expression vector was transformed into Escherichia coli BL21 (DE3), Rosetta (DE3), BL21 (DE3) pLysS bacterial strain respectively, and coated on a plate containing 50ug / mL kanamycin (Rosetta ( DE3), BL21(DE3)pLysS strains need to be supplemented with 50ug / mL chloramphenicol) for overnight culture at 37°C, pick a single colony and inoculate it into the liquid medium containing the corresponding antibiotics, culture overnight at 37°C, 200rpm, Sterilized 50% glycerol was added to the bacterial solution, mixed evenly and then frozen in a -80°C refrigerator to obtain a prokaryo...

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Abstract

The invention discloses a recombinant vector of a tea tree heat shock protein CssHSP-6 gene. The invention also discloses an expression method of the tea tree heat shock protein CssHSP-6 gene. A prokaryotic expression vector of the tea tree heat shock protein CssHSP-6 gene is constructed, a series of optimization is conducted on protein expression conditions, and finally a CssHSP-6 soluble proteinis obtained; the result lays a foundation for further studying the protein crystal structure and biological characteristics, and thus a basis is provided for improving the disease resistance of tea trees by a gene editing method.

Description

technical field [0001] The invention relates to a recombinant vector and an expression method of tea tree heat shock protein CssHSP-6 gene, belonging to the field of biotechnology. Background technique [0002] Tea tree, formerly known as: tea, a shrub or small tree of the family Theaceae, Camellia, with glabrous twigs. Leaves leathery, oblong or elliptic. The leaves of the tea tree can be used to make tea (different from the tea tree), the seeds can be used to extract oil, the material of the tea tree is fine and dense, and its wood can be used for carving. The distribution is mainly concentrated between 16 degrees south latitude and 30 degrees north latitude. Tea trees like warm and humid climates. The buds start to germinate when the average temperature is above 10°C, and the optimum temperature for growth is 20-25°C; the annual precipitation should be above 1000 mm; Sun-loving and shade-tolerant, it is suitable for growing under diffuse light; its life is divided into ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/70C12P21/02C07K14/415
CPCC12N15/66C12N15/70C12P21/02C07K14/415
Inventor 谢鑫李向阳蒋君梅任明见
Owner GUIZHOU UNIV
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