低毒病毒CHV1-EP721基因组序列的测定及侵染性全长cDNA克隆的构建
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摘要
低毒病毒是一类使板栗疫病菌毒力降低的无衣壳双链RNA病毒，携带低毒病毒CHV1-EP721的野生菌株EP721表现出典型的与低毒力相关的表型症状：色素分泌减少、菌丝体呈白色、产孢量降低、毒力下降等；但是与其它低毒病毒相比，CHV1-EP721在宿主中的累积量明显减少。本研究通过逆转录构建了CHV1-EP721的cDNA文库并测定了其全基因组序列。序列对比分析显示：CHV1-EP721与已测序的低毒病毒CHV1-Euro7和CHV1-EP713相比在核苷酸水平上的同源性平均为99％和90％；氨基酸水平上的同源性平均为98％和92％。用合适的内切酶辅助，精确连接全基因组的cDNA片段构建了CHV1-EP721具有侵染性的全长cDNA克隆p721。p721的体外转录产物成功转染无病毒的板栗疫病菌野生菌株EP721(-)、EP155和Euro7(-)。检测CHV1-EP721对不同宿主表型的影响以及在不同宿主中的累积量，结果显示感染了CHV1-EP721的不同宿主的表型相似，都和野生带毒菌株EP721表型一样菌体呈白色，可以产生少量分生孢子；并且在不同的宿主中CHV1-EP721的累积量没有明显的区别。这表明病毒累积量少是
广西大学硕士论文
CHVI一EP721病毒本身决定的。CHVI一EP721的侵染性克隆的获得，为
进行影响低毒病毒累积最的病毒基因的分析以及病毒与宿主相互作用的
研究提供了条件。
Hypoviruses are a group of dsRNA viruses associated with the reduced virulence (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica. The field strain EP721 of C. parasitica had been found to be infected by a hypovirus termed CHV1-EP721. EP721 shows typical hypovirulence-associated phenotype, reduced pigmentation, reduced asexual sporulation and female infertility. Comparison with other known hypoviruses, the accumulation of CHV1-EP721 in the cells of fungus is significantly lower. By construction and sequencing of a cDNA library of the virus, the whole genome sequence of CHV1-EP721 was determined. The virus showed extensive identities with the other two known hypovirus: CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at nuceotide level and 99% and 92% identities at amino acids level respectively.
The full-lenghth cDNA clone of the virus was constructed by accurately joining viral cDNA fragments according to the whole genome sequence with aid of restriction enzymes. By transfection, CHV1-EP721 was successfully introduced into virus-free fungal host strains EP721(-), EP155 and Euro7(-). All of the transfected strains showed similar phenotype to EP721 and the CHV1-EP721 dsRNA accumulation in different hosts were lower than those tranfected by CHV1-Euro7 and CHV1-EP713, conforming the low level of viral accumulation was the property of the virus. The availability of infectious cDNA clone of CHV1-EP721 makes it possible to dissect the molecular basis of a viral genome that controls the viral accumulation in the host and to understand other aspects of interaction between host and virus.
广西大学硕士论文
CHVI一EP721病毒本身决定的。CHVI一EP721的侵染性克隆的获得，为
进行影响低毒病毒累积最的病毒基因的分析以及病毒与宿主相互作用的
研究提供了条件。
Hypoviruses are a group of dsRNA viruses associated with the reduced virulence (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica. The field strain EP721 of C. parasitica had been found to be infected by a hypovirus termed CHV1-EP721. EP721 shows typical hypovirulence-associated phenotype, reduced pigmentation, reduced asexual sporulation and female infertility. Comparison with other known hypoviruses, the accumulation of CHV1-EP721 in the cells of fungus is significantly lower. By construction and sequencing of a cDNA library of the virus, the whole genome sequence of CHV1-EP721 was determined. The virus showed extensive identities with the other two known hypovirus: CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at nuceotide level and 99% and 92% identities at amino acids level respectively.
The full-lenghth cDNA clone of the virus was constructed by accurately joining viral cDNA fragments according to the whole genome sequence with aid of restriction enzymes. By transfection, CHV1-EP721 was successfully introduced into virus-free fungal host strains EP721(-), EP155 and Euro7(-). All of the transfected strains showed similar phenotype to EP721 and the CHV1-EP721 dsRNA accumulation in different hosts were lower than those tranfected by CHV1-Euro7 and CHV1-EP713, conforming the low level of viral accumulation was the property of the virus. The availability of infectious cDNA clone of CHV1-EP721 makes it possible to dissect the molecular basis of a viral genome that controls the viral accumulation in the host and to understand other aspects of interaction between host and virus.
引文
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3．徐耀先，周晓峰，刘立德，分子病毒学，武汉，湖北科学技术出版社，2000，11-30．
4. Wicker RB. 1993. Double-stranded RNA virus replication and packaging(minireview). The Jounal of Biological Chemistry 268(6):3797-3800.
5. Jcobs BL. and Langland JO. 1996. When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology 219:339-349.
6. Gitlin L. and Andino R. 2003. Nucleic acid -based immune systeme: the antiviral potential of mammalian RNA silencing. J. Virol. 77:7159-7165.
7. Goldbach R., Bucher E. and Prins M. 2003 Resistence mechanisms to plant viruses: an overview.Virus Res. 92:207-212.
8. Chandran K. and Nibert M.L. 2003. Animal cell invasion by large nonenveloped viruses: reovirus delivers the goods. Trends Microbiol. 11:374-382.
9. Hollings M. 1962. Viruses associated with a die-back disease of cultivated mushroom. Nature(London), 169: 692—695.
10. Lampson GP., Tytell AA. and Field AK. et al. 1967. Inducers of interferon and host resistance. Ⅰ. Double-stranded RNA from extracts of Penicillium funiculosum. Proc Natl Acad Sci U S A. 58(2): 782-9.
11．裘维蕃，菌物学大全，北京，科学出版社，1998，549-594．
12. Bevan EA., Herring AJ.and Midchell DJ. 1973. Prelimminary characterization of two species of dsRNA in yeast and their relationship to the "killer" character. Nature 245:81-86.
13. Herring AJ.and Bevan AE. 1974. Virus-like particles associated with the double-stranded RNA species found in killer and sensitive strains of the yeast Saccharomyces cerevisiae. J. Gen. Virol. 22: 387-394.
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15. Grente J. 1965. Les formes hypovirulentes d'Endothia parasitica et les espoirs de lutte contre le chancre du chataignier.C.R. Acad. Agric. France 51: 1033-1036.
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17. Castanho B. and Bulter EE. 1978. Rhizoctonia decline: A degenerative disease of Rhizoctonia solani. Phytopathol. 68:1505.
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