Ternary shuttle vector and method for establishing CLBV (Citrus Leaf Blotch Virus) infectious cloning with same

A technology of shuttle vectors and vectors, applied in the field of molecular biology, can solve the problems of difficulty in obtaining CLBV invasive clones, time-consuming and labor-intensive success rate, etc., to improve the acquisition rate of invasive clones, overcome toxicity or instability, and speed fast effect

Inactive Publication Date: 2018-09-21
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large genome of CLBV, it is time-consuming and labor-intensive with a low success rate to construct its invasive clones by using the traditional enzyme-cut ligation method
In previous attempts, it was difficult to obtain invasive clones of CLBV using Escherichia coli as a host, which may be related to the aforementioned instability

Method used

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  • Ternary shuttle vector and method for establishing CLBV (Citrus Leaf Blotch Virus) infectious cloning with same
  • Ternary shuttle vector and method for establishing CLBV (Citrus Leaf Blotch Virus) infectious cloning with same
  • Ternary shuttle vector and method for establishing CLBV (Citrus Leaf Blotch Virus) infectious cloning with same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of the ternary shuttle vector pCY

[0034] Firstly, the binary vector plasmid DK1317-2 was constructed: it was obtained by transforming plasmids PCMBIA1301 and pXT1. PCMBIA1301 was purchased from the market, and the pXT1 vector was donated by Professor Tao Xiaorong of Nanjing Agricultural University. Using the pXT1 vector as a template, primers TL1310F / TL1310R were used to amplify the fragment containing the pXT1 gene expression module (LB-2x35S-MCS-HDVRZ-NOS-RB). The pCAMBIA1301 plasmid was digested with Pvul. The amplified and digested products containing the expected fragments were then purified and fusion recombined using a gel extraction kit. The resulting fusion plasmid was digested with VspI, and the large fragments were reconnected to construct pCAMBI-2x35S-MCS-HDVRZ-NOS, namely DK1317-2.

[0035] The binary vector plasmid DK1317-2 was single-digested with restriction endonuclease Sac II to obtain a linearized vector. Enzyme digestion r...

Embodiment 2

[0038] Embodiment 2 constructs the invasive clone of CLBV

[0039] 1. Extraction of total RNA from susceptible leaves and segmental amplification of CLBV

[0040] Total RNA was extracted from susceptible leaves according to the instructions of Trizol reagent, and the integrity of RNA was detected by agarose gel electrophoresis. Using the extracted total RNA as a template, use PrimeScript TM II 1st Strand cDNA Synthesis Kit, synthesizes the first strand of cDNA. Using the cDNA as a template, two pairs of specific primers pCY-CLBV1F, CLBV1R and CLBV2F, pCY-CLBV2R were used for PCR amplification respectively, and two specific fragments CLBV1 and CLBV2 covering the entire length of the CLBV genome were obtained, with sizes of 4500bp and 4247bp respectively . The PCR reaction system is 25 μL, including: 8.5 μL of double distilled water, 12.5 μL of PrimerSTAR Max Premix (2×), 1 μL of specific upstream and downstream primers, and 1 μL of template. Reaction conditions: 98°C for 1m...

Embodiment 3

[0058] Example 3 Shuttle vector pCY is used for PVX invasive clone construction

[0059] Referring to the method of Example 2, the shuttle vector pCY of the present invention was used for PVX (Potato Virus X) invasive clone construction, the amplification of PVX full-length cDNA used primers pCY-PVXF, pCY-PVXR, and the yeast colony PCR detection used primers PVX-F, PVX-R. Transform the positive pCY-PVX plasmid into Agrobacterium C58C1 and infiltrate Nicotiana benthamiana through Agrobacterium injection. After 10 days of inoculation, the observation results show that the symptoms of tobacco injected with pCY-PVX are exactly the same as those of the positive control, and the symptoms of the tobacco injected with pCY-PVX are obviously the same as those of the negative control. comparison (such as Figure 5 shown). This shows that PVX has been highly expressed, and the yeast recombinant cloning system based on the three-way shuttle vector pCY has been successfully constructed, w...

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Abstract

The invention discloses a ternary shuttle vector pCY which has a nucleotide sequence of SEQ ID NO:17 as shown in the specification. The invention further discloses a method for establishing CLBV (Citrus Leaf Blotch Virus) infectious cloning. The method comprises the following steps: (1) extracting total RNA (Ribonucleic Acid) of an infectious CLBV plant, carrying out reverse transcription, and carrying out PCR (Polymerase Chain Reaction) amplification with pCY-CLBV1F, CLBV1R and CLBV2F and pCY-CLBV2R respectively so as to obtain specific segments CLBV1 and CLBV2 covering the whole length of aCLBV genome; (2) carrying out digestion on pCY with Stu I and Sma I; (3) carrying out TAR (Homologous Recombination) cloning, namely carrying out co-transformation on yeast YPH501 with CLBV1, CLBV2 and linearized pCY by using a lithium acetate method, and carrying out homologous recombination so as to obtain CLBV whole length cDNA (Complementary Deoxyribonucleic Acid) cloned pCY-CLBV; (4) carryingout pCY-CLBV plasmid transformation on agrobacterium, inoculating with citruses or nicotiana benthamiana, and carrying out identification, thereby completing CLBV infectious cloning.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a three-way shuttle vector and a method for constructing CLBV invasive clones using it. Background technique [0002] Citrus leaf blotch virus (CLBV) is a representative member of the genus Citrivirus in the Betaflexiviridae family, and can infect most citrus varieties by grafting and can also be transmitted by seeds , has a certain risk of spreading epidemics. The construction of CLBV invasive clones will help to understand its molecular characteristics and pathogenic mechanism, and also play an important role in the prevention and control of CLBV epidemics. In addition, CLBV does not cause obvious virus infection symptoms in most citrus varieties, and is expected to be developed as a virus vector with broad application prospects. The CLBV genome is about 8.7kb in size and contains three open reading frames (Open reading frame, ORF), which encode replication-related proteins, mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/70C12N15/66
CPCC12N15/70C12N15/8205
Inventor 宋震崔甜甜宾羽晏建红李中安周常勇
Owner SOUTHWEST UNIV
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