Pleurotus eryngii carboxin resistance screening and transforming vector as well as construction method and application thereof

A resistance screening and construction method technology, applied in the field of molecular biology, can solve the problem of high cost of gene point mutations and achieve good application prospects

Inactive Publication Date: 2018-12-21
SHANGHAI ACAD OF AGRI SCI
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a Pleurotus eryngii resistance screening transformation vector and its construction method and application, which solves the problem that the cost of point mutation of the gene is too high

Method used

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  • Pleurotus eryngii carboxin resistance screening and transforming vector as well as construction method and application thereof
  • Pleurotus eryngii carboxin resistance screening and transforming vector as well as construction method and application thereof
  • Pleurotus eryngii carboxin resistance screening and transforming vector as well as construction method and application thereof

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Embodiment 1

[0023] 1. Construction of Pleurotus eryngii wilting spirit resistance transformation vector:

[0024] (1) Extract the whole genome DNA of Pleurotus eryngii monokaryotic hyphae, and detect the DNA quality and concentration;

[0025] (2) A pair of complementary primers sdi2F and sdi1R ( figure 1 ); take the extracted genomic DNA as a template, utilize forward primer sdi1F and reverse primer sdi1R to amplify the first part of the DNA fragment (2251bp, shown in SEQ NO.7); take the extracted genomic DNA as a template, utilize forward Primer sdi2F and reverse primer sdi2R amplify the second part of the DNA fragment (339bp, as shown in SEQ NO.8); utilize the restriction endonuclease MfeI to digest the two parts of the DNA fragment, and then use T4 ligase to digest the two Partial DNA fragments are connected into a complete DNA fragment (2570bp, as shown in SEQ NO.9);

[0026] (3) The complete DNA fragment was ligated with a TA vector and introduced into the pGEM-Teasy plasmid vect...

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Abstract

The invention relates to a pleurotus eryngii carboxin resistance screening and transforming vector as well as a construction method and application thereof. A DNA fragment of succinate dehydrogenase iron sulfur subunit with point mutation is ligated into a plasmid vector, and is denoted as pTSdi. The transforming vector provided by the invention can be used for transforming pleurotus eryngii so asto enable the pleurotus eryngii to obtain the resistance to a fungicide-carboxin; in addition, an enzyme cleavage site for inserting an exogenous gene fragment is reserved in the constructed transforming vector, so that an interested exogenous gene can be conveniently inserted into the transforming vector and transformed into the pleurotus eryngii for performing genetic research; therefore, the pleurotus eryngii carboxin resistance screening and transforming vector has a good application prospect.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a Pleurotus eryngii wilt-resistant screening transformation vector and its construction method and application. Background technique [0002] In recent years, the industrialized cultivation of Pleurotus eryngii in my country has developed rapidly, and has become the fastest-growing edible fungus species in facility cultivation after Flammulina velutipes. Utilizing modern molecular biology techniques to improve Pleurotus eryngii varieties to obtain strains with high yield, short growth cycle and strong resistance to miscellaneous bacteria is an important topic in the genetic research of Pleurotus eryngii. To construct the genetic transformation system of Pleurotus eryngii, we must first establish molecular biology tools, such as resistance selection markers and promoters that drive the expression of foreign genes. [0003] Usually, point mutations in genes require the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/66C12N1/15C12R1/645
CPCC12N9/001C12N15/80C12Y103/99001
Inventor 尚俊军鲍大鹏李燕李焱杨瑞恒汪滢茅文俊唐利华
Owner SHANGHAI ACAD OF AGRI SCI
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