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Nimbya alternantherae effector Na2-g9900, and protein and application thereof

A technology of na2-g9900 and Alternaria, which is applied in the fields of application, herbicide and algicide, fungi, etc., can solve the problems such as the decrease of sporulation and pathogenicity of the strain, production obstacles, and lack of relevant work. , to achieve the effect of significant pathogenicity and good application prospects

Active Publication Date: 2018-08-24
ZHONGKAI UNIV OF AGRI & ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Alternaria pseudoalpacia has become an important biocontrol fungus of A. japonicus, but long-term storage or subculture will lead to a significant decrease in the sporulation and pathogenicity of the strain
Moreover, at present, domestic and foreign studies mainly focus on the physiological and biochemical research of Alternaria pseudoalpacia on the control of Asteroides phylloxera, but no related work has been carried out on the molecular mechanism of its toxin synthesis pathway.
However, the pathogenicity, toxin-producing ability and genetic stability of different mutant strains are often quite different, which will bring obstacles to future production

Method used

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  • Nimbya alternantherae effector Na2-g9900, and protein and application thereof
  • Nimbya alternantherae effector Na2-g9900, and protein and application thereof
  • Nimbya alternantherae effector Na2-g9900, and protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Alternaria pseudoalpacia effector Na2-g9900 gene cloning

[0038] In this study, the whole genome of Alternaria pseudoalpacia was sequenced, and a specific inducible expression effector g9900 was obtained, and then g9900 was cloned to construct its eukaryotic expression vector, which was introduced into yeast for inducible expression . Further research on its role in the pathogenic process of pathogenic bacteria.

[0039] The g9900 gene cloning method is as follows:

[0040] 1. Primer design

[0041] According to the g9900 sequence of the whole genome of Alternaria pseudoalpacia determined earlier, specific primers were designed as follows:

[0042] Primer g9900-F (shown in SEQ ID NO.4): gtgGAATTCatgaagtcctacgctgtcctc

[0043] Primer g9900-R (shown in SEQ ID NO.5): cggTCTAGAGagcagaagccaggcggc

[0044] 2. Gene cloning

[0045] Alternaria pseudoalpacia was cultured with PD medium, the mycelium was collected, and the total DNA and total RNA were extracted ...

Embodiment 2

[0050] Example 2 Eukaryotic expression of effector Na2-g9900

[0051] 1. Construction of eukaryotic expression vector

[0052] (1) Construction strategy: Digest the cloning vector and the expression vector containing the target fragment, and then connect and transform to construct the expression vector.

[0053] The eukaryotic expression vector pPICZαA used (such as figure 2Shown) is a special shuttle vector for eukaryotic expression in Pichia pastoris. After the target fragment is inserted, it can coincide with the reading frame of the vector to ensure that the triplet codon reads correctly during transcription and translation. The schematic diagram of the method of constructing the vector is shown in image 3 shown.

[0054] (2) Construction method

[0055] right Na2-g9900 Introduce EcoRI and XbaI enzyme cutting sites, use these two enzymes to digest pMD19-T-g9900 and eukaryotic expression vector pPICZαA respectively, recover the target fragments and connect them, tran...

Embodiment 3

[0063] Example 3 Effector Na2-g9900 expression product in vitro inoculation

[0064] 1. Express the effector Na2-g9900 according to the method in Example 2, take 10 μl of the eukaryotic expression product for 72 hours, and inoculate it on the flat and moist-cultured leaves of A. chinensis, observe the disease and take pictures to inoculate the empty vector pPICZαA expression product and The leaves of A. japonicus in sterile water were used as a control, and 3 repetitions were set.

[0065] 2. The result is as follows Figure 8 As shown, it shows that the expression protein of the effector Na2-g9900 can cause the pathogenicity of the leaves of A. japonicus, has significant pathogenicity to A. japonicus, is an important pathogenic effect factor, and has potential in the control of A. japonicus. Application prospect.

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Abstract

The invention discloses a Nimbya alternantherae effector Na2-g9900, and a protein and an application thereof. The nucleotide full-length sequence of the effector gene is represented by SEQ ID NO.1, the open reading frame sequence is represented by SEQ ID NO.2, and the amino acid sequence of the effector Na2-g9900 protein is represented by SEQ ID NO.3. The effector Na2-g9900 found IN the inventionhas significant pathogenicity to Alternanthera Philoxeroides, can be used for preparing preparation products for controlling Alternanthera Philoxeroides, can also be used for constructing biocontrol bacteria, and has an excellent application prospect in the treatment of Alternanthera philoxeroides.

Description

technical field [0001] The invention belongs to the technical field of pest management. More specifically, it relates to a Alternaria pseudoalpacia effector Na2-g9900 And its protein and application. Background technique [0002] Because of its rapid reproduction and strong adaptability, Alternanthera Philoxeroides (Mart.) Griseb. was introduced into Jiangsu and Zhejiang in the 1950s as a livestock feed plant, and then spread to all parts of the country. And cause weed damage, compete with crops for water, fertilizer, sunlight and growth space, resulting in severe crop yield reduction, and at the same time have extremely adverse effects on aquatic products, water conservancy, shipping, species resources, ecological environment, etc. It is one of the key targets of pest management in my country to control and kill malignant weeds in water areas. [0003] At present, in my country, chemical control is mainly used for the control of A. japonicus. There are several herbicides, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/37A01N47/44A01P13/00C12N15/81C12N1/19C12Q1/6895C12R1/84
CPCA01N47/44C07K14/37C12N15/815C12Q1/6895
Inventor 董章勇向梅梅肖永欣罗梅张云霞
Owner ZHONGKAI UNIV OF AGRI & ENG
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