Cloning Of DNA Components Of Banana Bunchy Top Virus Hainan Isolate And Prokaryotic Expression Of Coat Protein Gene

Banana bunchy top disease is one of serious diseases in banana, the pathogens of the disease is banana bunchy top virus.The virus has small isometric virions of 18-20nm.The genome consists of at least six components of circular ssDNA about 1.0~1.1kb. We designed six pairs of primers in accordance with the reported BBTV isolates. DNA 1-6 of BBTV in Hainan were amplified by PCR using total DNA from banana tissues which are of characteristic of typical BBTV symptom as template. The sequence analysis indicated that the six fragments are all full-length components of BBTV, the six fragments are 1104nt 1067nt 1059nt 1045nt 1014nt 1081nt respectively in length. All of them have been submitted to GenBank. All kinds of isolates from different countries were compared through the software of DNAsisst and clustalxa 83. Results showed that the sequence of component 1 is more conservative than other components , while both the sequences of component 2 and component 4 have more difference .Comparing component 1 in Hainan with other isolates ,we believe certainly that BBTV Hainan isolate belongs to Asia sub-group.Furthermore, CP gene of BBTV was amplified by PCR according to the sequence of DNA3, The CP gene is 528 nucleotides longth and it was inserted into the prokaryotic expression vector pET-22b(+) and pET-30a(+).Both digestion identification and sequencing demonstrated that the two recombinant vectors pET-22b-CP and pET-30a-CP have been correctly constructed. The two recombinant vectors were transformed into E.coli Rosetta(DE3) and BL21 ( DE3 ) plysS respectively.Transformants were cultured and induced by addition of isothiopropylgalactoside(IPTG) to a final concentration of 1mM. SDS-PAGE showed that the CP gene with pET-22b vector has been expressed in Rosetta(DE3) and CP withpET-30a vector has been expressed in BL21 (DE3) plysS. After 6 hours and 8 hours induction of IPTG respectively, the amount of the expression yield reached maximum, the amount of the expression yield of CP accounting for the total protein of the cells were 18.8% and 15.8% respectively. This study lay a firm foundation not only for preparation of antiserum later and detection BBTV fast and effectively, but also for plant anti-virus gene engineer and anti-virus breeding.