Amino acid sequence, gene sequence and expression vector of heat shock protein HmHSP 70 of hypsizygus marmoreus

A gene sequence, heat shock protein technology, applied in the field of molecular biology

Inactive Publication Date: 2009-10-21
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, people have a relatively deep understanding of heat shock proteins in plants and animals. There are few reports on heat shock proteins in microorganisms, and there are no reports on heat shock proteins in edible fungi. Genes can improve the tolerance of shimeji mushrooms and cope with sudden changes in temperature during the production process, which can relieve the restrictions on people's cultivation and production of shimeji mushrooms and lay a good foundation for the establishment of a microbial heat shock protein research platform

Method used

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  • Amino acid sequence, gene sequence and expression vector of heat shock protein HmHSP 70 of hypsizygus marmoreus
  • Amino acid sequence, gene sequence and expression vector of heat shock protein HmHSP 70 of hypsizygus marmoreus
  • Amino acid sequence, gene sequence and expression vector of heat shock protein HmHSP 70 of hypsizygus marmoreus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1: Cloning the middle fragment of the HmHSP70 gene of jiji mushroom

[0115] Step 1. Inoculate the female species (spores) of Pleurotus jimeji mushroom on the PDA plate, cultivate it at 25°C until the plate is full, and collect the mycelium. Using the CTAB method to extract genomic DNA, referring to the method of Shangguan Zhoujian et al. (Shangguan Zhoujian, Xie Baogui, Luo Lianzhong, etc. RAPD analysis of three selected strains of jiji mushrooms [J]. Chinese Edible Fungi, 2004, 3(23) : 12~14), and save it for future use.

[0116] Step 1. Design degenerate primers

[0117] Log in to the GenBank / DDBJ / EMBL database, and use Primer Premier5.0 to combine DNAMAN6. 0 program to design a pair of degenerate primers:

[0118] P1: 5'-GCTGTHRTYACHGTYCCAGCTTAYTTC-3'

[0119] P2: 5'-RGCDACRGCYTCRTCNGGRTTRAT-3'

[0120] The above primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the primers were diluted to 20 μmol L with sterile double-distill...

Embodiment 2

[0171] Example 2. Cloning of fragments at both ends of the HmHSP70 gene of Shimeji mushroom.

[0172] Step 1. Design specific primers

[0173] According to the sequencing result Seq ID No 1, use Primer Premier5.0 combined with DNAMAN6.0 program to design specific primers for both end fragments of the HmHSP70 gene:

[0174] P 3 : 5'-CAACGACAGGCTACTAAGGATGCTGG-3'

[0175] P 4 : 5'-ACACGGGGCACACGAGTCATACC-3'

[0176] The above primers were synthesized by Shanghai Sangon Bioengineering Service Technology Co., Ltd., and the primers were diluted to 20 μmol L with sterile double-distilled water before use. -1 .

[0177] Step 2 Extraction of total RNA from Shijiji mushroom

[0178] Total RNA was extracted by Trizol method. The operation is as follows.

[0179] 1. Preparation before RNA extraction

[0180] The pipette tip, EP tube, mortar, spoon and pestle used for RNA extraction should be processed before RNA extraction. All the utensils used in the experiment were treated w...

Embodiment 3

[0245] Example 3. Amplification of the Complete Reading Frame of the Mushroom HmHSP70 Gene

[0246] Step 1 Design full-length gene-specific primers to amplify the full-length gene

[0247] Using DNAMAN software to analyze the sequencing results of 3'RACE and 5'RACE products, splicing into the full sequence of HSP70, removing the linker sequence added by the RACE reaction, and according to the overlapping sequence of the 5' and 3' ends, a full-length splicing of the HSP70 gene was obtained The sequence of 2308bp is shown as SEQ ID NO4; the reading frame analysis of the full-length cDNA sequence was carried out by using the ORF finder software of NCBI. 2001bp, such as SEQ ID NO 7, encoding 667 amino acids, as shown in SEQ ID NO6.

[0248] According to SEQ ID NO4, use Primer Premier5.0 combined with DNAMAN6.0 program to design full-length specific primers to amplify the complete reading frame of the gene.

[0249] The primer design sequence is:

[0250] HSPORF1:5'-CCC AAGCTT...

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Abstract

The invention relates to an amino acid sequence, a gene sequence and an expression vector of a heat shock protein HmHSP70 of hypsizygus marmoreus, and belongs to the field of molecular biology. A complete open reading frame of the heat shock protein is acquired by RACE-PCR technology, and the amino acid sequence coded by the open reading frame is speculated; prokaryotic expression of the gene in colon bacillus makes the colon bacillus obtain obvious heat resistance so as to prove that the obtained gene sequence is genes of the heat shock protein; and the acquired gene provides new element for researching the heat shock protein, and the gene can be applied to transgene engineering to make a host transplanted into the gene obtain the heat resistance.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an amino acid sequence, a gene sequence and an expression vector of a heat shock protein HSP70 of P. Background technique [0002] Hypsizygus marmoreus is a rare edible fungus, which belongs to the medium-to-low temperature type edible fungus, and it occurs more frequently in late autumn and early spring under natural conditions. Mycelia growth temperature is 5-30°C, the most suitable is 20-25°C, mycelium will no longer grow when it exceeds 35°C or below 4°C, and it cannot survive above 45°C. The formation of primordia needs to be stimulated at a lower temperature of 10-16°C. The ideal temperature for fruiting body growth is 13-18°C. In particular, the temperature should be well controlled when buds are urged. When the temperature is too high (>25°C) or too low (<5°C), the fruiting should be temporarily stopped. When the temperature is too high, the mushroom cap becomes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/375C12N15/31C12N15/11C12N15/63C12N5/10C12N1/00C12N1/21C12R1/19
Inventor 郭立忠李翠翠卢伟东姜辉徐丽丽
Owner QINGDAO AGRI UNIV
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