Adenylate cyclase containing FYVE structural domain as well as coding gene and application thereof

A technology of adenylate cyclase and coding gene, which is applied in the field of pathogenic oomycete novel adenylate cyclase and its coding gene and application, and can solve the problems of soybean seed rot, economic loss, rot, etc.

Active Publication Date: 2020-09-11
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It infects soybeans from the roots to the stems. It can grow under wet conditions and in dense or heavy clay soils. It can cause soybean seed rot, seedling lodging, and rhizome withering and rot. In the field, it can cause serious damage. economic loss

Method used

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  • Adenylate cyclase containing FYVE structural domain as well as coding gene and application thereof
  • Adenylate cyclase containing FYVE structural domain as well as coding gene and application thereof
  • Adenylate cyclase containing FYVE structural domain as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1, Cloning of the adenylyl cyclase PsZFAC gene containing the FYVE domain in Phytophthora sojae

[0057] 1.1 Phytophthora soybean total RNA extraction

[0058] Phytophthora sojae strain P6497 was cultivated in the dark at 25°C for 4 days on 10% V8 solid medium, and the bacteria cake with a diameter of 5 mm was picked from the edge of the colony and inoculated in 10% V8 liquid medium, and harvested after 4 days of dark culture at 25°C For mycelium, take about 30mg of mycelium, put it into a 2mL mold-free and sterile centrifuge tube, add two steel balls with a diameter of 5mm, freeze it in liquid nitrogen, and use a ball mill to grind it to powder. The SV Total RNA Extraction Kit (Z3100) from Promega Company was used to extract Phytophthora sojae RNA, and the specific method steps refer to the description of plant tissue RNA extraction in the kit.

[0059] 1.2 Synthesis of first-strand cDNA by reverse transcription of Phytophthora sojae

[0060] First-strand cDN...

Embodiment 2

[0066] Example 2, Analysis of the expression pattern of PsZFAC gene in different developmental stages of Phytophthora soybean

[0067] Selected kit: Takara TB Green TMPremix Ex Taq TM II (Tli RNase H Plus) (Code: RR820A). Dilute 0, 4, 4 with mycelial stage cDNA samples 2 、4 3 、4 4 、4 5 、4 6 times, using PsActin (see SEQ ID No.13 and SEQ ID No.14 for primer sequences) as an internal reference gene and PsZFAC as a detection gene, a real-time fluorescent quantitative PCR standard curve was established. The amplification efficiency of gene primers needs to be above 90%, and an appropriate diluted sample concentration is selected for the next test.

[0068] Samples diluted 4 times at different developmental stages and infection stages were used as templates, PsActin was used as an internal reference gene, and real-time fluorescence was performed using detection primers PsZFAC-qPCR F / PsZFAC-qPCR (see SEQ ID No.5 and SEQ ID No.6 for the sequence) Quantitative PCR detection. ...

Embodiment 3

[0070] Embodiment 3, the acquisition of the knockout transformant of Phytophthora sojae PsZFAC gene

[0071] 3.1 Construction of the knockout vector of Phytophthora sojae PsZFAC gene

[0072] (1) Obtain candidate sgRNA

[0073] The sgRNA of PsZFAC was designed by EuPaGDT (http: / / grna.ctegd.uga.edu / ), and the full-length genome was searched on the website.

[0074] (2) Secondary structure screening

[0075] Use website tools

[0076] (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html) Analyze the secondary structure of candidate sgRNAs, select the number of hairpin complementary bridges that is less than or equal to 3 as candidate sgRNAs.

[0077] (3) Off-target analysis

[0078] The off-target analysis of sgRNA was carried out through FungiDB (www.fungidb.org), and a total of 23 bases including the PAM region were input for comparison, including 9-23 (including NGG) complete matches, and the risk of off-target was considered to be high.

[0079]...

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Abstract

The invention discloses adenylate cyclase containing an FYVE structural domain from phytophthora sojae as well as an encoding gene and application of the adenylate cyclase. The adenylate cyclase is prepared from phytophthora sojae. The adenylate cyclase PsZFAC containing the FYVE structural domain has an amino acid sequence as shown in SEQ ID No. 4, and a coding gene of the adenylate cyclase PsZFAC has a nucleotide sequence as shown in SEQ ID No. 3. The main function of the PsZFAC protein is to control the formation of phytophthora sojae sporangium. The gene provided by the invention has a very high application value in controlling phytophthora sojae root rot, and a bactericide developed by taking the protein as an action target has important practical significance in controlling the occurrence and prevalence of phytophthora sojae root rot.

Description

technical field [0001] The present invention relates to a novel adenylate cyclase of pathogenic oomycete and its encoding gene and application, in particular to the adenylate cyclase PsZFAC containing FYVE from Phytophthora soybean and its encoding gene and application. Background technique [0002] Phytophthora sojae is one of the important soil-borne plant pathogenic oomycetes. It was first known to the world in North America in the 1950s. Its host range is very narrow, and the only crop it can infect is soybean. It infects soybeans from the roots to the stems. It can grow under wet conditions and in dense or heavy clay soils. It can cause soybean seed rot, seedling lodging, and rhizome withering and rot. In the field, it can cause serious damage. economic loss. [0003] Phytophthora sojae is a semi-parasitic vegetative oomycete. Asexual reproduction usually produces three types of asexual spores: sporangia, zoospores and chlamydospores; the sporangia are pear-shaped, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N5/10C12N15/90C12N1/15C12R1/645
CPCC12N9/88C12N15/902C12Y406/01001
Inventor 刘西莉苗建强杜晓冉张晋辉金朵刘小飞高虎虎李文浩汝冰璐
Owner NORTHWEST A & F UNIV
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