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Coded gene of cysteine proteinase of cotton and application

A cysteine ​​protease, coding technology, applied in the field of recombination and functional analysis and application, cloning of cotton cysteine ​​protease gene

Inactive Publication Date: 2006-04-19
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene has not yet been cloned in cotton

Method used

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  • Coded gene of cysteine proteinase of cotton and application
  • Coded gene of cysteine proteinase of cotton and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1: the cloning method of coding cotton cysteine ​​protease gene:

[0077] 1 Extraction of total RNA

[0078] The solution for extracting RNA must be treated with DEPC. Add 0.1% DEPC to the double-distilled water used to prepare the mother solution, stir and mix overnight, and sterilize under high pressure for use. Use ethanol and isopropanol immediately after opening. All glassware was baked at 2000C for 6 hours, plastic products were soaked in 0.1% DEPC solution for 30 minutes, and then sterilized by high pressure to inhibit the activity of nuclease before use.

[0079] (1) 12 ml of extract (200mmol L-1 Tis-HCL, Ph 8.5, 300mmol L-1, 10mmol L-1EDTA, 1.5% sodium deoxycholate,

[0080] 1.5% Nonidep-40) was mixed with 120 microliters of mercaptoethanol and 1% PVP (W / V), added to a centrifuge tube, and cooled in an ice-water bath.

[0081] (2) Take about 1 gram of the material, grind it into powder with liquid nitrogen, transfer it into a centrifuge tube, mix i...

Embodiment 2

[0123] Embodiment 2: Cotton cysteine ​​protease gene Ghcysp sequence

[0124] The results of the cloned gene sequence analysis are as follows:

[0125] (1) Information of SEQ.IN.NO 1

[0126] (a) Sequential features

[0127] Length: 1368 base pairs

[0128] Type: nucleic acid

[0129] Chain type: double chain

[0130] Topology: Linear

[0131] (b) Molecular type: cDNA

[0132] (c) Assumption: No

[0133] (d) Antisense: no

[0134] (e) Original source: Cotton

[0135] (f) Sequence description: SEQ.IN.NO 1

[0136] 1 ATTACCAATA CACATCAAAC TTTTTCACTA TAAAACCCCA CTTCAAAACC CTTTTGGAGT

[0137] 61 AATCAAATTA GGATCTAATC CTTCAACTTT CTAAACCAAT GGCTTTGCTG CAAATTTTTC

[0138] 121 TCTTCGTTGC TCTGGTTCTA TCATTCTGTTTTTCCATCCAACTTGCTGGA CTGTCTCGTC

[0139] 181 CACTCTTGGA TGAAGACTCC ATGAGGCATG AGGAGTGGAT GAGTCAACAT GGCCGTGTTT

[0140] 241 ACGCGGATGA GCAGGAGGAC CATAAGAACA AGCGCTTCAA TGTGTTTAAA GAGAACGTCG

[0141] 301 AACGAATTGA AGAGTTTAAT GACGGAAAAA CTTTCAAACT TGCGATAAAT CAGTTTGCT...

Embodiment 3

[0173] Embodiment 3: Construction of expression vector

[0174] 1 According to the coding cotton cysteine ​​protease gene Ghcysp sequence, design the following PCR primers:

[0175] Forward primer: 5'AAAACC CCA CTT CAA AAC CC 3'

[0176] Reverse primer: 5'GCA CAC GAT TCA TTC ATAAC 3'

[0177] The cDNA of senescent leaves was used as a template for RT-PCR amplification to obtain PCR products. 2 Take 2 μl of the PCR product and connect it with pGEM-T Vector. The method is carried out according to the instructions of the Promega product. The connection product is transformed into DH5α bacteria (purchased from Shanghai Sangong Bioengineering Co., Ltd.), and the transformed bacteria are on LB / ampicillin / IPTG / -Gal solid Medium culture, 37 ℃ inverted culture for 12-20 hours, some colonies are white, some become blue. Select the white colony, extract the plasmid DNA, carry out enzyme digestion and PCR identification.

[0178] 3 Cut out the fragment with a restriction endonuclease,...

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Abstract

A gene for coding the cysteine proteinase of cotton is cloned and recombined through taking the cDNA as template from the senile leaf of cotton, designing primer, PCR, recovering the amplified DNA fragment, linking it with p GEM-T carrier, transferring it to colibacillus, screening recon, analyzing sequence, fast amplifying to obtain full-length cDNA, configuring anti-sense expression carrier, transferring to Arabidopsis, and comparing to prove its delayed sanility. It can be used to culture the transgenic crops for preventing early sanility.

Description

【Technical field】 [0001] The invention relates to the cloning, recombination, function analysis and application of cotton cysteine ​​protease gene (Ghcysp), and belongs to the field of molecular biology and biotechnology. 【Background technique】 [0002] In cotton production, premature senescence of cotton leaves is common. Premature senescence of cotton leaves generally leads to a yield loss of about 10%, and a cotton field with severe premature senescence loses more than 20%. The premature senescence of cotton leaves not only affects the yield of cotton, but also leads to the reduction of cotton fiber strength, shortened length, and decreased uniformity, thereby reducing the yarn quality of cotton fibers. Therefore, cotton production urgently needs technologies and methods for delaying cotton premature senility. [0003] Programmed cell death is a genetically controlled, highly ordered process of cell suicide, which enables organisms to remove aging cells. It is necessary f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N15/29C12N9/50C12N15/10C12N15/70C12N15/82
Inventor 喻树迅沈法富
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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