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Novel plant DNA virus carrier and its use

A DNA virus, a new type of plant technology, applied in the direction of plant genetic improvement, botanical equipment and methods, applications, etc., can solve problems such as laborious and time-consuming plant regeneration, low expression of foreign genes, and impact on the industrialization process of medicinal proteins.

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

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Problems solved by technology

However, the expression level 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 overall industry of using transgenic plants to produce medicinal proteins process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0037] Collect Chinese tomato yellow leaf curl virus samples, extract the total plant DNA, use this as a template, and use beta01(5'- GGTACC ACTACGCTACGCAGCAGCC-3', containing KpnI site) and heta02 (5'- GGTACC TACCCTCCCAGGGGTACAC-3', containing the KpnI site) was used as a primer to amplify the full-length genome of DNAβ by PCR, and the PCR product was purified by QIAquick Gel Extraction Kit and cloned into pGEM-T Easy Vector to obtain a full-length copy of the clone pGEM-T -β (New) and its sequence was determined, and the full-length genome sequence of DNA-β was 1.3kp, and the sequence was as No.1-12. The homology between Chinese tomato yellow leaf curl virus DNAβ is 72%-99%, and the homology with other reported DNAβ (AYVVβ, CLCuMVβ, CLCuRVβ and BYVMVβ) is only 35-43%. DNAβ is half the size of DNA-A, and has no sequence homology with all geminivirus DNA-A except for the highly conserved replication initiation sequence TAATATT / AC. Although the homology between DNA β is very ...

Embodiment 3

[0047] DNA-A must be present in the presence of DNAβ to cause disease, and DNAβ has only one gene C1 that is conserved in length and position, so mutation of the C1 gene will clarify the function of C1. The method for introducing mutant bases in primers of the present invention mutates the C1 gene of Y10 DNAβ, so that the gene cannot be transcribed. When this mutant was inoculated with Nicotiana benthamiana together with DNA-A, it did not show the typical symptoms of curved leaves. Further Southern Blot detection of the DNA of the leaves of the plants found that DNAβ was detected in plants without symptoms, indicating that after the mutation of the C1 gene No longer causing pathogenicity, but still able to replicate, indicating that the gene does play an important role in pathogenicity. Example 3 Construction of virus-induced gene silencing vector and establishment of method

[0048] After the C1 gene in DNA β is mutated, DNA β can still replicate, so if the C1 gene is replac...

Embodiment 4

[0057] 25-30 days after inoculation, the upper leaves of the inoculated plant begin to turn white along the veins, and gradually expand, so that all the upper leaves along the veins are bleached. After the plants are silent, they can last for a long time. The inoculated virus does not show any symptoms on the plant due to the deletion of the C1 gene, and the phenotype displayed on the host plant is the phenotype after the plant is induced to silence. Example 4 Construction of Plant Expression Vector and Establishment of System

[0058] The C1 gene of DNAβ is replaced by foreign protein, and the transcription and expression of the foreign gene are initiated by the promoter and terminator of the C1 gene during the replication of the virus. Geminivirus-based vectors require two forward-repeated genome sequences, so after the introduction of multiple cloning sites, foreign fragments still need to be inserted twice. Therefore, further transformations have been carried out on the ba...

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Abstract

The plant DNA virus carrier includes the infectious carrier of Chinese tomato yellowing curly leaf virus DNA-A and DNA-beta, derived plant gene silencing carrier and virus expressing carrier. The present invention also provides one new kind of satellite DNA molecule constructing plant DNA virus carrier and it has at least 72% homology with any one sequence of SEQ ID No.1-12. The present invention also provides the use and method of utilizing plant DNA virus carrier in virus functional genome research, plant functional genome research and foreign protein expression. The present invention provides one important platorm for researching virus genomee structure, function and expression and regulation mechanism and the interaction between virus and plant; provides one excellent tool for researching plant functional gene; and establishing one technological platform for using plant as bioreactor expressing foreign protein.

Description

technical field [0001] The present invention relates to a novel plant DNA virus vector and its application, specifically, the present invention relates to the infectious vector of Chinese tomato yellow leaf curl virus DNA-A and DNAβ and the plant gene silencing vector and virus expression vector derived from it . The invention also relates to a DNA beta satellite molecule for constructing a plant DNA virus vector and a whole genome sequence thereof. The invention also relates to an application and method of using the plant DNA virus vector to determine genome functions, plant genome functions and express foreign proteins. Background technique [0002] Whitefly-transmitted geminivirus (WTG) is a kind of plant single-stranded DNA virus that occurs widely in the world. loss. WTG belongs to the Begomovirus genus (Begomovirus) in the Geminiviridae family (Geminiviridae) in taxonomy, including more than 100 independent species. Under natural conditio...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/83C12Q1/68C12Q1/70
Inventor 周雪平陶小荣谢艳李正和李桂新
Owner ZHEJIANG UNIV
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