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Application of colletotrichum gloeosporioides fatty acid hydroxylase CsSCS7 and method for constructing gene knockout vector and gene knockout mutant

A fatty acid hydroxylase and gene knockout technology, applied in the biological field, can solve the problems such as failure to prove the sensitive regulation of SCS7, no research on the function of SCS7, etc., and achieve good application prospects and improve the effect of degradation.

Active Publication Date: 2021-01-29
HAINAN UNIVERSITY
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  • Abstract
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  • Application Information

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Problems solved by technology

[0004] Therefore, based on the fact that the existing studies failed to prove that SCS7 is involved in the regulation of bacterial sensitivity to drugs, there is currently no related functional research on SCS7 in anthrax bacteria

Method used

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  • Application of colletotrichum gloeosporioides fatty acid hydroxylase CsSCS7 and method for constructing gene knockout vector and gene knockout mutant
  • Application of colletotrichum gloeosporioides fatty acid hydroxylase CsSCS7 and method for constructing gene knockout vector and gene knockout mutant
  • Application of colletotrichum gloeosporioides fatty acid hydroxylase CsSCS7 and method for constructing gene knockout vector and gene knockout mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Cloning of embodiment 1-Hevea anthracis fatty acid hydroxylase CsSCS7 gene

[0032] The CsSCS7 gene sequence of anthracnose was obtained by searching in the NCBI database, and the homologous sequence was obtained by using the local BLAST method in the C. siamense HN08 transcriptome sequence database (preserved in our laboratory), and the primer pair CsSCS7-F( 5'-CGCGGATCCATGCCGTCGAGAACTCTCCCTA-3') / CsSCS7-R(5'-CCCAAGCTTTTATTGCGTCTTGACAATGGGAG-3'). The DNA and cDNA of C. siamense HN08 were respectively used as templates for amplification. According to sequence analysis, it was shown that the obtained sequence contained a complete coding open reading frame. The size of the DNA sequence is 1271bp, and the size of the cDNA sequence is 1215bp. The gene contains an intron, encodes 404 amino acids, contains a CytB5 domain, a low complexity region, a transmembrane region, a hydroxylase, and Yeast SCS7 is homologous, and the gene is named CsSCS7 gene, which is a fatty acid hydr...

Embodiment 2

[0033] Example 2-Construction of Bacillus anthracis Fatty Acid Hydroxylase CsSCS7 Gene Knockout Vector

[0034] The primer pairs CsSCS7-U-F / CsSCS7-U-R and CsSCS7-D-F / CsSCS7-D-R were designed before and after the reading frame of the fatty acid hydroxylase CsSCS7 gene of B. anthracis, and the upper arm sequence and the lower arm sequence after the C-terminal of the CsSCS7 gene were obtained by PCR amplification , using the method of homologous recombination, the upper arm and lower arm sequences were joined into the vector pCX62-S, and the knockout vector pCX62-S-CsSCS7 was obtained. For the schematic diagram, see figure 1 .

[0035] The sequence of primer CsSCS7-U-F is:

[0036] 5'-GTACCGGGCCCCCCCAGCTTCTCAGAATCCACATATCCAC-3'

[0037] The sequence of primer CsSCS7-U-R is:

[0038] 5'-CGATACCGTCGACCTCGAAGCTGCAGGTGGCGATCGTGAAT-3'

[0039] The sequence of primer CsSCS7-D-F is:

[0040] 5'-GCTCTCACCGCGGATCCTTCATCGACCAACGTTCATAC-3'

[0041] The sequence of primer CsSCS7-D-R is...

Embodiment 3

[0043] Example 3 - Construction of Bacillus anthracis Fatty Acid Hydroxylase CsSCS7 Gene Knockout Mutant

[0044] The knockout vector pCX62-S-CsSCS7 constructed and obtained in Example 2 was introduced into protoplasts of Hevea anthracnose (C.siamense) HN08 by using the PEG-mediated protoplast transformation method. )’s DCM medium for screening, transformed 6 batches in total, and obtained 57 transformants.

[0045]Genomic DNA sequences of 57 transformants were extracted in batches and verified by PCR. The transformant △CsSCS7-51 was in line with expectations. -3'), the wild type can amplify a target band with a size of about 1200bp, but the mutant does not amplify the target band, CsSCS7 gene upstream primer CsSCS7-Ou-F (5'-TGCTTTTGCATTGCTGTGACC-3') and chlorimuron-methyl-resistant gene ILV1 internal primer ILV-R (5'-GTTCAACGCCGCCTTCCGACAAAAT-3'), the mutant can amplify a target band with a size of about 2700bp, but the wild-type HN08 does not amplify the band; The chlorimu...

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Abstract

The invention provides an application of colletotrichum gloeosporioides fatty acid hydroxylase CsSCS7 and a method for constructing a gene knockout vector and a gene knockout mutant, and particularlyrelates to the application of the colletotrichum gloeosporioides fatty acid hydroxylase CsSCS7 in degrading bactericides, the CsSCS7 gene knockout mutant is obtained by constructing a CsSCS7 gene knockout vector, through functional tests, it is proved that the hydroxylase is extremely important for growth of colletotrichum gloeosporioides hyphae, participates in regulation and control of degradation of fungi to fludioxonil, can be applied to degradation of pyrrole sterilizing agents such as fludioxonil and has a good application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an application of an anthrax fatty acid hydroxylase CsSCS7 and a method for constructing a gene knockout vector and a gene knockout mutant. Background technique [0002] Sphingolipids (sphingolipids) are components of eukaryotic cell plasma membranes and widely exist in animals, plants and fungi. There are two complex sphingolipids in fungi: inositolphosphoceramides (IPC) and glucosylceramide (GlcCer). Experiments in plant pathogenic fungi have proved that these two types of complex sphingolipids are involved in the growth and pathogenic process of various fungi. For example, the inhibition of IPC synthase of Botrytis cinerea seriously affects spore germination and mycelium growth; the ceramide synthase FgBar1 in Fusarium graminearum is specifically connected to the synthesis of GlcCer, and the deletion or methylation modification of GlcCer will cause Loss of pathogenicity of head ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/80C12N1/15C12R1/645
CPCC12N9/0071C12N15/80
Inventor 林春花龙熙平缪卫国刘文波廖小淼李潇
Owner HAINAN UNIVERSITY
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