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Plant virus satellite DNA beta molecule, infectivity clone and method for preparing expression carrier

A DNA molecule and plant virus technology, applied in the field of genetic engineering, can solve the problems of low exogenous gene expression, laborious and time-consuming plant regeneration process, and affect the industrialization process, etc., to achieve fast proliferation, small genome, and low equipment requirements Effect

Inactive Publication Date: 2003-06-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of exogenous genes in transgenic plants is relatively low, generally less than 1% of the soluble protein, and the process of gene transformation and plant regeneration is laborious and time-consuming, which largely affects the comprehensive industrialization of the production of medicinal proteins using transgenic plants process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0028] The full-length DNA β of Chinese Saikui Yellow Vein Virus is 1348bp (GenBank accession number AJ421622), presumably encoding 8 ORFs in total, and contains a 115nt length between nucleotides 1249 and 15. All DNA β molecules are highly homologous and conserved Region (conserved region, CR) (SEQ ID NO.6), CR region contains the nonanucleotide sequence TAATATTAC shared with Saikui yellow vein virus DNA-A, it is speculated that this region is DNA-A coded replication protein recognition and interaction as a site. Saikui Huangvei virus DNAβ contains an A-rich region (SEQ ID NO.7) between nucleotides 767 and 971, and the nucleotide content is as high as 60.5% A. It is believed that the A-rich region is involved in the packaging process of DNAβ molecules associated with the size requirements. A 118aa-long C1 protein (SEQ ID NO.8) is encoded on the complementary strand of Saikui Yellowvein Virus DNAβ. After mutating the start codon ATG of C1ORF, the pathogenicity of the virus de...

Embodiment 3

[0031] The results showed that the complete DNAβ sequence of Saikui Yellow Vein Virus had the highest homology with Multan cotton leaf curl virus CLCuMVβ01 and CLCuMVβ02, which were 66.7% and 67%, respectively; followed by Rajasthan cotton leaf curl virus CLCuRVβ homology, which was 61.8%; The homology with the okra yellow vein mosaic virus BYVMVβ is 46%; the homology with the red thistle yellow vein virus AYVVβ, the Chinese tomato yellow leaf curl virus TYLCCNVY10β and the tobacco curly stem virus TCSVY2β are only 40.6% and 40.4%, respectively. % and 40.2%. It shows that the homology between DNAβ is very low. Example 3 Comparison of the nucleotide homology of the CR region of Saikui Huangvei virus DNAβ and other reported DNAβ molecules

[0032] With the race Kui yellow vein virus DNA β of the present invention and GenBank reported Sheng red thistle yellow vein virus (Ageratum yellow vein virus, AYVV β, AJ252072), okra yellow vein mosaic virus (Bhendi yellow vein mosaic virus...

Embodiment 4

[0036] Using the total plant DNA as a template, with SEQ ID NO.9 (5'-GGTCACTACGCTACGCAGCAGCC-3', without KpnI site) and SEQ ID NO.3 (5'- GGTACC TACCCTCCCAGGGGTACAC-3', containing the KpnI site) was amplified, and the PCR product was inserted into pGEM-T Easy Vector to obtain pGEM-T-β(2), which cloned another full-length genome copy. Digest pGEM-T-β(1) with KpnI to obtain a 1.3kb full-length genome fragment and insert it into the corresponding site of pGEM-T-β(2) to obtain pGEM-T-2β, and then use EcoRI at both ends of pGEM-TEasy Vector Two copies of DNAβ were excised at the site, inserted into the EcoRI site of the Agrobacterium vector pBINplus, and transformed into competent cells DH5α to obtain the DNAβ-infectious clone pBINplus-2β. Example 5 Construction of Saikui Yellow Vein Virus DNA-A Infectious Clones

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Abstract

The present invention discloses one plant virus satellite DNA beta molecule, its infections cloning and the preparation process of its expression carrier. The plant virus satellite DNA beta molecule is coromadel coast falsemallow yellow vein virus satellite DNA-beta, which has whole sequence length of 1348 bp, no homology with geminivirus DNA-A sequence, one sectino of 115 nt conserved area, one 118 and open reading frame and one A-rich area in different length. The present invention also relates to the infections cloning of the virus and the infectious cloning may be used in virus functional genome research. The present invention also provides the expression carrier constructing method and preparation process of the satellite DNA-beta.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a plant virus satellite DNA beta molecule, an infectious clone and a method for preparing an expression vector. More specifically, the present invention relates to a satellite DNA β of a whitefly-transmitted geminivirus Malvastrum coromandelisnum yellow vein virus (MYVV) and its entire genome sequence. The present invention also relates to a novel plant DNA infective clone, in particular, relates to the infective clone of the whitefly-transmitted geminivirus Saikui Huangvei virus, and the present invention also relates to the use of the infective clone to carry out viral functional genome research methods. The invention also relates to a plant DNA virus expression vector and a preparation method thereof, in particular to an expression vector and a preparation method of the whitefly-transmitted geminivirus Saikui yellow vein virus satellite DNAβ. ...

Claims

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Application Information

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IPC IPC(8): A01H1/00C07H21/04C12N15/34C12N15/82
Inventor 周雪平谢艳崔晓峰陶小荣李正和
Owner ZHEJIANG UNIV
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