Novel rapid preparation methods for cDNA dimer and infectious clone of citrus viroid

A virus-infectious, citrus-like technology, applied in the field of molecular biology, can solve the problems of time-consuming, cumbersome steps, high cost, etc., and achieve the unique effect of the method

Inactive Publication Date: 2017-10-24
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional virus-like construction method is cumbersome, time-consuming and l...

Method used

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  • Novel rapid preparation methods for cDNA dimer and infectious clone of citrus viroid
  • Novel rapid preparation methods for cDNA dimer and infectious clone of citrus viroid
  • Novel rapid preparation methods for cDNA dimer and infectious clone of citrus viroid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 class of virus resources and one-step RT-PCR

[0029] The CEVd virus source was taken from the navel orange 'Meishan 9', and the CBCVd virus source was taken from the miscellaneous mandarin orange 'Zhujian'. Total RNA from plant leaves was extracted using a plant RNA rapid extraction kit (or trizol method). Design specific primers using PrimeScript TMOne-step RT-PCR kit (Catalog No.: RR055A, TaKaRa Company, Dalian, China) directly amplifies the full-length sequence of the required viroid dimer. The CEVd-specific primer pair used in the present invention is CEVd-For (5'-ggaaacctggaggaagtcgag-3') and CEVd-Rev (5'-ccggggatccctgaaggactt-3'), and the CBCVd-specific primer pair is CBCVd-For:(5'- ggggaaatctcttcagac-3') and CBCVd-Rev (5'-ggggatccctcttcaggt-3').

[0030] Using the obtained total RNA to directly amplify CEVd by one-step RT-PCR, the specific reaction system and amplification conditions are as follows:

[0031] 1. CEVd reaction system:

[0032]

...

Embodiment 2

[0042] Example 2 Construction and in vitro transcription of CEVd / CBCVd dimer cloning vector

[0043] Purify the CEVd / CBCVd dimer obtained by one-step RT-PCR in Example 1 and clone it into the pGEM-Teasy vector to obtain a ligation product comprising CEVd / CBCVd dimer. The principle is as follows: figure 2 As shown, T7 promoter site and SpeI site were used to synthesize CEVd or CBCVd dimer RNA. Transform into competent cells Escherichia coli DH5α for amplification, and the bacterial liquid sequencing determines the dominant sequence and insertion direction of the required CEVd / CBCVd dimer. Extract the plasmid, and purify it with ethanol after linearizing the plasmid with the restriction endonuclease SpeI. Steps such as dimer purification, cloning, transformation competence, and plasmid linearization can be performed according to the operating instructions of relevant reagents.

[0044] Purify with ethanol after SpeI linearized the plasmid:

[0045] First, the obtained mixed ...

Embodiment 3

[0050] Example 3 In vitro transcription product activity verification

[0051] The CEVd / CBCVd dimer RNA obtained by in vitro transcription was diluted to 500ng / ul with diluent (100mM Tris-HCl, 10mM EDTA, pH 7.5), and the in vitro transcription products of CEVd and CBCVd were inoculated with the woody indicator plant citron and the The in vitro transcription product of CEVd was rubbed inoculated with the herbaceous host tomato, and the symptoms of the infected citron were observed after four months (results see image 3 ) and RT-PCR detection (results see Figure 4 ), and extract the total RNA from the leaves of the herbaceous host tomato, and use the digoxin-labeled probe method to carry out Northern blot experiments to further verify the expression activity of CEVd (results in Figure 5 ).

[0052] The results showed that the CEVd-inoculated citron showed typical symptoms of CEVd infection, and the northern hybridization signal of CEVd-infected tomato was obvious, and the r...

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Abstract

The invention discloses a novel rapid preparation method for a cDNA dimer of citrus viroid. The method includes the steps of: 1) extracting total RNA of citrus viroid; 2) through a one-step RT-PCR method, amplifying the total RNA, wherein a PCR reaction system includes PrimeScript 1 step Enzyme Mix, MgCl2, a viroid full-length amplification primer, the total RNA, and water without RNA enzyme, and after the amplification, the citrus viroid cDNA dimer is prepared. The invention also discloses a novel rapid preparation method for infectious clone of the citrus viroid. The method includes the steps of: producing the cDNA dimer of citrus viroid through the mentioned method, and cloning the dimer into a T-vector to form a linking product comprising the dimer, thus obtaining the infectious clone. The method is quick, high-effective and stable, has unique processes, and is a novel method for producing the cDNA dimer of citrus viroid.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a new method for rapid preparation of citrus virus cDNA dimer and its infectious clone. Background technique [0002] Viroids are small closed circular single-stranded RNAs with a size of 246-401-nt, which can cause serious diseases on sensitive hosts and are important plant pathogens. Citrus exocortis viroid (CEVd) belongs to the potato tuber viroid family (Pospiviridae) Pospiviroid genus (Pospiviroid), is the pathogen of citrus split skin disease, which can cause severe cracking on the rootstocks of orange and orange. Peel symptoms are one of the important diseases that threaten the development of the citrus industry in the world. Citrus bark cracking viroid (CBCVd) belongs to the family Pospiviroidae (Pospiviroidae) of the genus Cocadviroid and can co-infect citrange rootstocks with other citrus viroids in the absence of CEVd Causes symptoms similar to citrus split peel. Hop d...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/63C12R1/93
CPCC12N15/1096C12N15/63
Inventor 曹孟籍王亚飞周常勇
Owner SOUTHWEST UNIVERSITY
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