Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of aspartic protease gene in improvement of beauveria bassiana variety

A technology of Beauveria bassiana and aspartic acid, which is applied in the direction of plant genetic improvement, application, genetic engineering, etc., can solve problems such as unreported high temperature tolerance, achieve improved yield and virulence, and improve high temperature tolerance effect of ability

Active Publication Date: 2021-09-17
SOUTHWEST UNIVERSITY
View PDF19 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the research on high temperature tolerance of strains regulated by aspartic protease

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of aspartic protease gene in improvement of beauveria bassiana variety
  • Application of aspartic protease gene in improvement of beauveria bassiana variety
  • Application of aspartic protease gene in improvement of beauveria bassiana variety

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, BbASP gene cloning

[0036] Design upstream and downstream primers according to the aspartic protease sequence in NCBI:

[0037] ASP-F: 5'-ATGTCGCTCAGAAACATCGTC-3' (SEQ ID NO.1);

[0038] ASP-R: 5'-TTACTTGAGATTAGCGAAGCC-3' (SEQ ID NO.2);

[0039] Using the genomic DNA of wild-type Beauveria Bassiana Bb0062 as a template, use Max DNA Polymerase was used for PCR amplification, and the product was gel-recovered after gel electrophoresis to verify that the size was correct, and then the fragment was connected to the cloning vector pEASY-Blunt, and the BbASP gene was obtained after enzyme digestion and sequencing were verified to be correct.

[0040] The full length of BbASP genomic DNA is 1208bp (SEQ ID NO.3), containing two introns, the full length of ORF sequence is 1068bp (SEQ ID NO.4), encoding 355 amino acids (SEQ ID NO.5), and the predicted protein molecular weight is 37.5kDa.

Embodiment 2

[0041] Example 2, Expression analysis of BbASP gene under high temperature stress

[0042] Two culture methods A and B were used to detect the difference in the expression of BbASP gene in the conidia and hyphae of Beauveria bassiana under normal temperature (26°C) and high temperature (32°C) stress.

[0043] (A) Take 50μL concentration of 1×10 7 Conidia / mL wild-type spore suspension of Beauveria bassiana was placed on CZM solid medium, and cultured at 26°C and 32°C for 14 days respectively, then the conidia RNA was extracted and reverse-transcribed into cDNA, and the γ-Actin gene was used as the The expression level of BbASP gene was detected by internal standard, and no stress was used as the control;

[0044] (B) Take 50μL concentration of 1×10 7 Conidia / mL wild-type spore suspension of Beauveria bassiana was added to 1 / 4 SDY liquid medium, cultured on a shaker at 26°C and 200rpm for 3 days, then stressed at 32°C for 4 hours, and mycelial RNA was extracted and reverse tra...

Embodiment 3

[0051] Embodiment 3, construction of BbASP gene homologous knockout vector

[0052] Homologous knockout backbone vector selection pK 2 -PtrpC-bar-TtrpC (referred to as pK 2 -bar), which was developed by our lab in the backbone vector pK 2 Based on the transformation obtained, pK 2 -PtrpC-bar-TtrpC vector schematic diagram as figure 2 As shown, bar is a glufosinate-resistant gene, and the nucleotide sequence of the bar gene is shown in SEQ ID NO.10. A sequence was selected at the 5' end and 3' end of the ORF region of the BbASP gene as a homology arm, and primers were designed , the underline represents the enzyme cleavage site:

[0053] LB-F: 5'-CG GAATTC GCATGCTCACTTTGCAACTTC-3' (EcoRI, SEQ ID NO.11);

[0054] LB-R: 5'-CG GAATTC CGATGACTGACGCTGAATTG-3' (EcoRI, SEQ ID NO. 12);

[0055] RB-F: 5'-GC TCTAGA GGTCAAGTTTATCGCGACGTC-3' (XbaI, SEQ ID NO. 13);

[0056] RB-R: 5'-CCC AAGCTT CTTTGCAGCAATCCATGAGTC-3' (Hind III, SEQ ID NO. 14);

[0057] The fragments of the...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses application of an aspartic protease gene in improvement of beauveria bassiana variety. The yield and toxicity of conidia of the beauveria bassiana are improved by knocking out the aspartic protease gene BbASP, or the high-temperature tolerance of the beauveria bassiana is improved by over-expressing the aspartic protease gene BbASP, and the nucleotide sequence of the aspartic protease gene BbASP is as shown in SEQ ID NO. 20; if the BbASP knockout strain is cultured for 10 d at 26 DEG C, the conidium yield is 72.57% higher than that of a wild type, if the BbASP knockout strain is cultured for 20 d at 26 DEG C, the conidium yield is 59.43% higher than that of a wild type, the survival rate of a host is reduced, the half lethal time is shortened, the time that entomogenous thalli penetrate out of the dead host is shortened, and the growth rate of the BbASP over-expression strain is increased under the high-temperature stress of 32 DEG C; and after the conidia are stressed at the high temperature of 43 DEG C for 2 h and 4 h, the germination rates are respectively increased by 7% and 11.34%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of the aspartic acid protease gene in improving Beauveria bassiana species. Background technique [0002] my country is a traditional agricultural country, and the sustainable development of agriculture is inseparable from the use of pesticides. Chemical pesticides are widely used because of their good insecticidal effect, quick effect, and simple use. However, the abuse of chemical pesticides will cause environmental pollution, pesticide residues, poisoning by mistaken ingestion of humans and animals, and so on. The demand for toxic pesticides is increasing, and biological pesticides are one of the most effective alternatives. Beauveria bassiana is a broad-spectrum entomopathogenic fungus, which is easy to cultivate and has a wide pathogenic spectrum. It can infect more than 700 kinds of insects in 149 families of 15 orders. Because of its lethal effect on insects ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/57A01N63/30A01P7/04C12R1/645
CPCC12N9/58A01N63/30Y02A50/30
Inventor 金丹赵文琦宋代军李先碧张艳范艳华
Owner SOUTHWEST UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com