No.6 gene segment of rice dwarfing virus

A dwarf virus and gene fragment technology, which is applied in the field of molecular biology of plant viruses, can solve the problems of inability to obtain RDV plant reovirus infective clones, complex RDV genome structure, and inability to mechanically spread, etc. The effect of dwarf disease, extensive agricultural utilization value, general theory and application value

Inactive Publication Date: 2002-04-17
PEKING UNIV
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  • Summary
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AI Technical Summary

Problems solved by technology

Due to the complex structure of the RDV genome and the inability to transmit mechanically
Therefore, it is impossible to use antisense genetics to study the function of non-structural protein genes, such as intercellular motor proteins, in the complete plant reovirus genome, resulting in the fact that RDV and other plant reoviruses cannot be obtained at present. Infectious clones of viruses

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0016] Complementation experiments were carried out between the Potato virus X virus cell-to-cell motility-deficient strain pPVX25KmGUS containing the Gus reporter gene and each gene component in the RDV genome, and it was proved that the cell-to-cell movement of the motility-deficient virus strain pPVX25KmGUS in tobacco leaves can be achieved by co-expressing the RDVS6 gene It can be restored by complementation, while other gene components in the RDV genome do not have the above functions. In pPVX25KmGUS, the intercellular motor protein 25K contains a deletion mutation resulting in the deletion of 72 amino acids at the C-terminus, and cannot perform intercellular movement. The specific steps are:

[0017] (1) Construction of plant transient expression vector clone comprising RDV-S6 gene

[0018] The RDV-S6 gene was released using appropriate restriction enzymes (BamHI and PstI), and cloned into a plant transient expression vector with a CaMV 35S promoter. After antibiotic s...

example 2

[0021] Using green fluorescent protein (EGFP) as a reporter gene to transiently express the fusion gene of S6 gene and EGFP in epidermal cells of tobacco leaves, it was found that the protein encoded by RDV gene S6 (Pns6) was distributed in the plasmodesmata of the cell wall in a punctate manner, and in the cytoplasm form irregular aggregates and can enter the nucleus. The specific steps are:

[0022] (1) Acquisition of RDV S6-GFP fusion gene

[0023] The fusion gene of S6 and enhanced green fluorescent protein (EGFP, Clontech) was obtained by two-step recombinant PCR.

[0024] A. According to the base sequences of RDV S6 gene and EGFP gene, two pairs of primers were designed respectively:

[0025] S6 forward primer:

[0026] 5'AAA ATG GAC ACA GAA ACT CTT TGC-3'

[0027] S6 reverse primer:

[0028] 5’CC TCG CCC TTG CTC ACC ATT TTG TAC ACG GTA ATA GCA-3’

[0029] This primer contains the complementary sequence at the 3' end of the S6 gene and the starting sequence of the ...

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Abstract

The present invention relates to the biological function and determination method of non-structural protein gene S6 of rice dwarf virus (RDV). The non-structural protein Pns6 is encoded by the No.6 segment S6 of RDV. The infections cloning test of Potato Virus X intercellular motion defect strain and the complementary test of RDV encoded non-structural protein gene shows that non-structural protein encoded by RDV is intercellular motion protein of RDV. Intracellular location research of Pns6 shows that the S6 protein is located in the plasmodesmus of plant cell wall and has the typical characteristic of motion protein. The determination of the gene biological function of S6 in helpful to the further research of the plant infecting mechanism of RDV and obtaining more powerful RDV resisting plant.

Description

Technical field: [0001] The invention belongs to the field of molecular biology of plant viruses, in particular to the determination of the biological function of rice dwarf virus (RDV) non-structural protein gene S6. Background technique: [0002] Rice dwarf virus (RDV) is a member of the Reoviridae family and the plant reovirus genus with a double-stranded RNA genome. It causes severe rice diseases in Southeast Asia and causes severe economic losses. [0003] Rice dwarf virus (RDV) genome consists of 12 double-stranded RNAs, the total length of the genome is 25617bp, encoding 7 structural proteins (P1, P2, P3, P5, P7, P8 and P8') and at least 6 non-structural proteins proteins (Pns4, Pns6, Pns9, Pns10, Pns11 and Pns12). The function of these nonstructural proteins is unclear. Due to the complex structure of the RDV genome, it cannot be transmitted mechanically. Therefore, it is impossible to use antisense genetics to study the functions of non-structural protein genes, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/40C12N15/46C12Q1/68
Inventor 李毅魏春红吴刚刘慧君
Owner PEKING UNIV
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