Soybean disease-green related virus infectious cloning vector and efficient soybean inoculation method

A technology of virus infectivity and cloning vectors, which is applied in the field of soybean disease-related virus-infectious cloning vectors and high-efficiency inoculation of soybeans, and can solve the problems of soybeans that are not easy to infect

Pending Publication Date: 2022-07-29
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] The present invention constructs the invasive clone of soybean cyanosis-associated virus, and can successfully infect soybean through hairy roots induced by Agrobacterium rhizogenes K599, and provides a solution to the infectivity of soybean geminivirus (soybean cyanosis-associated virus) A method for cloning less susceptible soybeans

Method used

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  • Soybean disease-green related virus infectious cloning vector and efficient soybean inoculation method
  • Soybean disease-green related virus infectious cloning vector and efficient soybean inoculation method
  • Soybean disease-green related virus infectious cloning vector and efficient soybean inoculation method

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Embodiment 1

[0040] Soybean Zhengqing-related virus was isolated from the soybean samples suspected of being infected by geminiviruses collected in the field of Yuanyang County, Henan Province, and its complete genome sequence is shown in the accession number MT221447 (shown in SEQ ID NO.1). ). The 2.0 tandem repeats of soybean disease green related virus (SoSGV) whole genome sequence was constructed into pBINPLUS vector to construct recombinant vector pBINPLUS-2.0×SoSGV, and the constructed soybean disease green related virus infectious cloning vector pBINPLUS-2.0×SoSGV Introduced into Agrobacterium rhizogenes K599, K599 carried SoSGV-infectious clones. A method for inoculation of soybean gemini virus mediated by Agrobacterium rhizogenes K599 was established to achieve successful systemic infection of the virus. Application of screening SoSGV-resistant soybean varieties and mining of SoSGV-resistant gene resources.

[0041] The process of constructing the 2.0 tandemly repeated SoSGV gen...

Embodiment 2

[0048] Using Agrobacterium rhizogenes K599 to inoculate Wanhua 518 varieties, the steps are as follows:

[0049] 1. The successful pBINPLUS-2.0×SoSGV electric shock constructed and verified in Example 1 was introduced into Agrobacterium K599;

[0050] 2. Optimization of Agrobacterium treatment solution

[0051] In sterile water, prepare the treatment solution at the following concentrations:

[0052] [2-(4-morpholino)ethanesulfonic acid]: 0.5g / L

[0053] Sucrose: 20g / L

[0054] B5: 3.21g / L

[0055] Acetosyringone: 100 μM.

[0056] 3. Use the Agrobacterium treatment solution to adjust the OD600 of A. rhizogenes with pBINPLUS-2.0×SoSGV to 1.0.

[0057] 4. Induced systemic infection: The hypocotyls of Wanhua 518 soybean seedlings germinated for 7 days were excised, the oblique sections were coated with bacterial cells, and placed in sterile vermiculite; in addition, 5 mL of treated hairy roots were used for each plant. Watered with Agrobacterium liquid, sealed and moisturis...

Embodiment 3

[0062] Using Agrobacterium rhizogenes K599 to inoculate the Williams 82 variety, the steps are as follows:

[0063] 1. The successfully verified pBINPLUS-2.0×SoSGV was electroporated into Agrobacterium K599;

[0064] 2. Optimization of Agrobacterium treatment solution

[0065] In sterile water, prepare the treatment solution at the following concentrations:

[0066] [2-(4-morpholino)ethanesulfonic acid]: 0.5g / L

[0067] Sucrose: 20g / L

[0068] B5: 3.21g / L

[0069] Acetosyringone: 100 μM

[0070] 3. Use the Agrobacterium treatment solution to adjust the OD600 of A. rhizogenes with pBINPLUS-2.0×SoSGV to 1.0.

[0071] 4. Induced systemic infection: The hypocotyls of Williams 82 soybean seedlings germinated for 7 days were excised, the oblique sections were coated with bacterial cells, and placed in sterile vermiculite; in addition, 5 mL of treated hair was used for each plant. The Agrobacterium tumefaciens solution was irrigated, and rooting was induced after 2 weeks of air...

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Abstract

The invention discloses an infectious cloning vector of soybean disease-green related virus and a high-efficiency soybean inoculation method, and the infectious cloning vector of soybean disease-green related virus (SoSGV) is prepared by constructing not less than 1.2 tandem and repeated whole genome sequences of soybean disease-green related virus on a pBINPLUS vector; the soybean can be successfully infected by the hairy roots induced by the agrobacterium rhizogenes K599, and a method for solving the problem that the soybean is not easily infected by the soybean geminivirus (soybean green related virus) infectious clone is provided. According to the invention, only three primers are used for constructing 2.0 tandem repeat infectious cloning vectors; pCR and Western Blot detection results show that the K599-induced soybean disease-green related virus can realize systematic infection in a plurality of soybean varieties to generate disease-green symptoms, and the K599-induced soybean disease-green related virus can be used as a subsequent screening resistance variety system.

Description

technical field [0001] The invention relates to the field of plant virology and genetic engineering, in particular to an infectious cloning vector of soybean disease green related virus (SoSGV) and an efficient soybean inoculation method. Background technique [0002] Soybean "Zongqing" refers to the phenomenon that when soybeans are normally mature, the plants or some branches are still leafy and green, and there are pods but the pods are empty or the grains are shriveled. Through Small RNA sequencing combined with bioinformatics analysis, technicians identified a new recombinant gemini virus from diseased soybean plants in many places in the main soybean producing area of ​​Huanghuaihai, which was tentatively named as soybean disease green related virus (Soybean stay- green associated virus, SoSGV) (Cheng, R. et al. A new distinctgeminivirus causes soybean stay-green disease. Molecular Plant (2022) doi:10 / gptbfj). [0003] Geminiviridae viruses are a class of plant DNA vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/34C12N15/66C12N1/21A01H5/00A01H6/54A01G7/06A01G22/40C12R1/01
CPCC12N15/8205C12N15/8283C12N15/66C07K14/005A01G7/06A01G22/40C12N2750/12022
Inventor 陶小荣徐毅程锐祥梅若鑫
Owner NANJING AGRICULTURAL UNIVERSITY
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