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FsCYP51 gene and application thereof

A technology of gene and gene coding, applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effect of reducing pathogenicity

Active Publication Date: 2021-10-08
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the research on the CYP51 gene of Fusarium sugarcane

Method used

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  • FsCYP51 gene and application thereof
  • FsCYP51 gene and application thereof
  • FsCYP51 gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Extraction of Genomic DNA and Total RNA, Synthesis of cDNA and Full-length Cloning of Fusarium Saccharum FsCYP51 Gene and CDS

[0032]Genomic DNA of Fusarium sugarcane was extracted by SDS method. Total RNA of Fusarium saccharum was extracted by Trizol method. The purity and concentration of the extracted DNA and RNA were detected by 1% agarose gel electrophoresis and ultra-micro ultraviolet spectrophotometer (Nanodrop). cDNA synthesis was performed using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNAwiper) reverse transcription kit (Nanjing Novizan Biotechnology), and the reaction system and procedures were carried out according to the kit instructions. The extracted genomic DNA was used for full-length gene cloning, and the cDNA obtained by reverse transcription was used for full-length CDS cloning.

[0033] Table 1 Primer Sequence

[0034]

[0035] Note: The sequence of lowercase letters indicates the recognition site of restriction endonuclease...

Embodiment 2

[0038] Example 2 Bioinformatics analysis of FsCYP51 gene

[0039] Use the online analysis tool ProtParam (https: / / web.expasy.org / protparam / ) to analyze the FsCYP51 protein sequence; use the online software GSDS2.0 (http: / / gsds.gao-lab.org / ) to analyze the FsCYP51 gene Analysis of the structure; use NCBI CDD to analyze the conserved functional domain of FsCYP51 protein; use the online software SignalP-5.0Server to analyze the signal peptide of FsCYP51 protein; use the online analysis software NetPhos 3.1Server (http: / / www.cbs.dtu.dk / services / NetPhos / ) to predict the potential phosphorylation modification sites of FsCYP51 protein; through the online software SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma. html) analyze the secondary structure of FsCYP51 protein; use the online software Phyre2 (http: / / www.sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index) to predict the tertiary structure of FsCYP51 protein; use The online tool TMHMM Server v2.0 (h...

Embodiment 3

[0050] Example 3 Construction of FsCYP51 Gene HIGS Plant Expression Vector and Biolistic-mediated Genetic Transformation of Sugarcane with HiGS Expression Vector

[0051]According to the cloned CDS sequence and full-length gene sequence of FsCYP51, select two specific segments of the CDS of the FsCYP51 gene and a segment of the UTR region (as shown in SEQ NO: 4 to SEQ NO: 6, wherein, SEQ NO: 6 is the UTR region fragment), and these three segments were concatenated into one fragment as the HIGS target fragment, and the concatenated fragment was synthesized by Shanghai Sangong. Combined with the enzyme cutting sites on the RNAi plant expression vector, Primer Premier 5 was used to design primers tgtf-F / R, tgtr- For F / R and loop-F / R (Table 1), PCR was performed using the synthesized target fragment as a template, and the corresponding target fragment was recovered with a gel recovery kit after 1.5% agarose gel electrophoresis. The vector pBWA(V)BU was digested with restriction e...

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Abstract

The invention relates to the technical field of bioengineering, in particular to an FsCYP51 gene and an application thereof. According to the FsCYP51 gene and the application thereof. An FsCYP51 gene full length and a CDS full length are obtained by using Fusarium sacchari as a test material and designing a specific primer and cloning the specific primer according to genome sequencing data. Meanwhile, a multivalent HIGS plant expression vector is constructed according to the cloned FsCYP51 gene full length and CDS full length, and an interference fragment is successfully transformed into a sugarcane receptor material by utilizing a gene gun mediated genetic transformation method, so that a foundation is laid for researching the FsCYP51 gene function and creating the tip rot resistant sugarcane germplasm.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a FsCYP51 gene and its application. Background technique [0002] Sugarcane (Saccharum officinarum L.) belongs to the grass family Saccharum and is the most important sugar crop in my country. Pokkah boeng disease is a fungal disease that is widely distributed in the world. Fusarium sacchari is one of the main pathogens of sugarcane tip rot in China. In recent years, due to the continuous planting of sugarcane and the large-scale promotion of susceptible varieties such as Xintai sugar series, the sugarcane tip rot in my country has shown an outbreak trend, which has seriously affected the production of sugarcane and the economic development of planting areas. One of the major diseases of sugarcane. Therefore, the prevention and control of sugarcane tip rot will be an urgent problem for the sugarcane industry. [0003] Sterol 14α-demethylase (Sterol 14α-demethylase, P450...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82A01H5/00A01H6/46
CPCC12N9/0073C12N15/8282C12Y114/1307
Inventor 姚伟张木清周宇明黄振段真珍徐世强暴怡雪
Owner GUANGXI UNIV
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