Transformation Of NtBT1 Gene Into Nicotiana Tabacum And Cultivation Of Anti-PVY Transgenic

Tobacco is one of the important economic crops in our country.The main application of tobacco is the harvest of tobacco leaves for consumers to taste,by industrial processing into a variety of tobacco products.As a result,the quality of tobacco is very important.Compared with the tobacco products with good quality,bad the taste of tobacco productsis very poor,besides,the tar and nicotine in tobacco products are much more,which creates a severe impact to the consumers' lungs.Tobacco virus disease is one of the severe tobacco common virus occur in the tobacco growing areas in the world.The types of tobacco virus disease is numerous,which are widely distributed.Since the 1950s,the tobacco virus disease around the world began to arise,which caused great loss in tobacco production.Potato PVY is one of the virus in potato virus Y group(potyvirus).PVY virus can infect many kinds of plants,such as Solanaceae,Chenopodiaceae and leguminous plants.Tobacco potato Y virus disease is caused by potato virus Y(PVY)infection of tobacco which lead to the a systemic disease.PVY disease is also called "vein necrosis disease" and "vein banding disease".The main sympotoms of PVY infection are always mosaic,necrosis and leaf rolling.Early infection of the virus leads to low yield,and infection of this virus nearly harvest tobacco,always affect the quality of tobacco products.With the aim of mining more PVY resistant genes from plant genome used in plant molecular breeding,transcriptome of tobacco infected by PVY was sequenced in previous study.We have found a gene which was significantly up-regulated by PVY treatment.Sequence analysis showed that this gene contained two typical structure domains:BTB domain and TAZ domain.This gene belongs to the BTB-TAZ(BT)family,and designated as NtBT1(Nicotiana tabacum BTB and TAZ domain protein 1).In this study,NtBT1 gene was tranformed into tobacco main cultivar "K326" using the agrobacterium mediated method.We have got a plenty of transgenic plants after molecular biological test.After screening on growth medium supplemental of Km,several stransgenic tobacco lines were selected with NtBTl gene stably inherited.After anti-virus detection of the seedlings from T1 transgenic lines,the anti-virus transgenic tobacco lines will be obtained.These transgenic lines will be used for subsequent breeding of new tobacco cultivar and further mechanism analysis of anti-virus mediated by NtBTl gene.The main results were showed as follows:(1)NtBTl gene was extracted from Nicotiana tabacum.Expression levels analysis showed that NtBT1 transcript level was induced by PVY treatment,and the expression levels in different tissues and different growth period were different.The plant expression vector pBHT-BT1 was constructed using construct pBHT-005 and NtBT1 gene adding SfiI restriction sites.(2)NtBTl gene was transformed into tobacco cultivar "K326" using the agrobacterium mediated method.In total.After twice PCR detection,32 PCR positive plants were got.After RT-PCR detection,30 RT-PCR positive plants were obtained for further analysis.(3)7 plants were randomly selected for the Southern Blot analysis,results showed that the 3 plants were transgenic positive plants.(4)Now we got 4 transgenic lines,after Km screening combined with PCR detection to T1 generation of tobacco transgenic lines,the PCR testing positive for get 5 strains of T1 generation of transgenic plants.(5)Using the T1 generation of tobacco transgenic lines and K326 wild-type do the experiments of antiviral test,the experimental results show that the T1 generation of tobacco transgenic lines compared with wild type to PVYN virus has obvious resistance.