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Application of diaphorina citri ubiquitin-binding enzyme E2J2 gene in prevention and control of citrus huanglongbing

A technology of ubiquitin-conjugating enzyme and citrus Huanglongbing, which is applied in the application field of the citrus psyllid ubiquitin-conjugating enzyme E2J2 gene in the prevention and control of citrus Huanglongbing, which can solve the difficulties in the prevention and control of Huanglongbing, which plagues the citrus industry and biodiversity Destruction and other issues

Active Publication Date: 2022-07-15
GANNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, Huanglongbing bacteria cannot be artificially cultivated, but they can grow and reproduce in citrus psyllids for a long time. Once citrus psyllids are infected by Huanglongbing bacteria (CLas), they can carry and spread rapidly for life. It has brought great difficulties and is the biggest problem plaguing the citrus industry in recent years
The current control of Huanglongbing in the international citrus industry is mainly limited to the use of chemical pesticides to control the occurrence of citrus psyllids. However, the frequent use of chemical pesticides has caused pesticide residues, environmental pollution, biodiversity destruction and pest resistance. and many other issues

Method used

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  • Application of diaphorina citri ubiquitin-binding enzyme E2J2 gene in prevention and control of citrus huanglongbing
  • Application of diaphorina citri ubiquitin-binding enzyme E2J2 gene in prevention and control of citrus huanglongbing
  • Application of diaphorina citri ubiquitin-binding enzyme E2J2 gene in prevention and control of citrus huanglongbing

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Experimental program
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Effect test

Embodiment 1

[0037] The cDNA sequence of the citrus psyllid ubiquitin-conjugating enzyme E2J2 gene (DcUbcE2J2) shown in SEQ ID NO.1 was entrusted to Nanjing GenScript Biotechnology Co., Ltd. for prokaryotic expression and purification. The purified protein was obtained and verified by Western Blot. The results are as follows figure 1 shown.

Embodiment 2

[0039] The purified protein obtained in Example 1 was subjected to an in vitro ubiquitination binding experiment to detect whether the citrus psyllid ubiquitin-conjugating enzyme E2J2 gene (DcUbcE2J2) of the present invention has ubiquitin-binding activity. The experimental steps are as follows:

[0040] The constructed in vitro ubiquitination reaction system is shown in Table 1:

[0041] Table 1 Constructed in vitro ubiquitination reaction system

[0042]

[0043] The role of imidazole in Table 1 is to prevent the binding of His-tagged E1 and E2 to the nickel-agarose column.

[0044] Since as a ubiquitin-conjugating enzyme, the activation of ubiquitin-activating enzyme E1 is required to achieve the binding function, so the present invention designs two groups of experiments under the same conditions of in vitro ubiquitination liquid environment, the difference lies in whether E1 is activated. Specifically, ①E1+E2+Ub; ②E2+Ub.

[0045]Insert the EP tube containing the rea...

Embodiment 3

[0047] dsRNA synthesis and RNAi experiment

[0048] (1) dsRNA synthesis

[0049] Taking citrus psyllid adult cDNA as a template, by PCR technology, using the amplification primers PT-F: 5'ATTCAAAACCACCAAGCATCT3', PT-R: 5'TCTTCTTGTGCTCGTCTGTCT 3' as shown in SEQ ID NO.3 and SEQ ID NO.4 A partial fragment of the E2J2 gene was obtained, and the amplified product was recovered with the Omega gel recovery kit; the amplified product was ligated into the pMD19-t plasmid, transformed into E. The LB liquid medium was expanded and shaken, and the bacterial solution was sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing; the bacterial solution with low mutation rate was selected for expansion and the plasmid was extracted (Sigma Rapid Small Plasmid Extraction Kit); using the plasmid as the template, Using primers containing T7 promoters with nucleotide sequences as shown in SEQ ID NO.5 and SEQ ID NO.6, RNAi-F: TAATACGACTCACTATAGGG ATTCAAACCACCAAGCATCT, RNAi-R: TAATACGAC...

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Abstract

The invention provides application of diaphorina citri ubiquitin-binding enzyme E2J2 gene in prevention and control of citrus huanglongbing, and belongs to the technical field of animal gene engineering. The invention provides the application of the diaphorina citri ubiquitin-binding enzyme E2J2 gene with a nucleotide sequence as shown in SEQ ID NO.1 in prevention and control of the citrus huanglongbing. A specific primer is designed to synthesize dsRNA, the silent E2J2 gene is interfered by RNA, the gene silencing efficiency of the gene is detected, the gene is subsequently placed on a branch infected with Candidatus Liberibacter asiaticum for feeding, and the influence of the gene on Candidatus Liberibacter asiaticum (CLas) infection is studied. By blocking the transmission path of the liberobacter asiaticum in the diaphorina citri instead of controlling the diaphorina citri, a new strategy is provided for effective prevention and control of the liberobacter asiaticum, and a new prevention and control thought is provided for controlling transmission of bacterial pathogens by means of vector insects.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to the application of the citrus psyllid ubiquitin-conjugating enzyme E2J2 gene in the prevention and control of citrus Huanglongbing. Background technique [0002] Citrus Huanglongbing (HLB) is one of the most devastating diseases in the world's citrus industry. Since there is currently no effective treatment for susceptible trees, Huanglongbing is also known as the "cancer" of citrus. So far, the huanglongbing pathogen cannot be artificially cultivated, but it can grow and reproduce in the citrus psyllid for a long time. Once the citrus psyllid is infected by CLas, it can be carried for life and spread rapidly, which is helpful for the study of huanglongbing disease and its prevention and control. It has brought huge difficulties and is the biggest problem that has plagued the citrus industry in recent years. At present, the control of Huanglongbin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/87A01G13/00
CPCC12N9/104C12N15/87A01G13/00Y02A50/30
Inventor 卢占军曾祥东张金波余海中方瑜婕胡威陈玮黄爱军彭婷姚锋先邹小金谢廉颉汪和贵舒佳旺刘淑婷
Owner GANNAN NORMAL UNIV
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