Rapid Construction of
Full-length cDNA Clones of Tobacco Mosaic Virus and the Infectivity Assay of
Its in Vitro Transcript
XUE Chao-Yang, ZHOU Xue-Ping*, CHEN Qing, QI
Yi-Jun, CHAO Hai-Feng, LI De-Bao
( Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China
)
Abstract The
full-length cDNA of tobacco mosaic virus faba bean isolate (TMV-B) was
amplified with RT-PCR in which T7 promoter sequence was added in the 5′
terminus of its upstream primer, so that full-length cDNA was put directly
under the control of a T7 promoter. The cDNA was cloned into plasmid pT7Blue
and linear DNA was got by digesting the recombinant with KpnI or KpnI and PstI. Using these
linear DNA and full-length PCR product as templates, respectively, their in vitro transcripts
were inoculated to Nicotiana
tabacum
and Chenopodium
amaranticolor. All of the transcripts had infectivity and produced
symptoms similar to that of wild TMV. It was found that transcripts of the full-length
PCR product had higher infectious efficiency than those of linear DNA.
Key words tobacco mosaic
virus； full-length infectious cDNA； long template PCR
Received:
November 8, 1999 Accepted: January 3, 2000
This work was supported by Special Grant for Younger Qualified
Scientists from Natural Science Foundation of Zhejiang Province and Ministry of
Education
* Corresponding author: Tel, 86-571-6971680; Fax,
86-571-6961525; e-mail, [email protected]