Study On Safety Transgenic Tobacco Toward Breeding Varieties With High Resistance To Bacterial Wilt Disease

Tobacco is an important economic crop in China.Bacterial wilt disease caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases of tobacco,which is the key limiting factor for the yield and quality of tobacco.There are not any efficient chemical methods or other methods to control it so far.Developing disease-resistant tobacco cultivars is an efficient strategy to solve bacterial wilt.But there are some problems in the traditional disease-resistant cross-breeding,such as long cycle breeding,negative correlation between resistance and high yield and quality and so on.The effective way to solve R.solanacearum infection is breeding high yield and quality tobacco varieties with resistance to bacterial wilt disease through molecular breeding technology.Based on available root-specific genes and promoter,terminator was cloned form tobacco and construct self-deleted of antibiotics vectors.Expression vectors harboring disease resistance gene driven by specific promoters were constructed and were transformed into tobacco for functional identification.The function of specific promoter deletion was researched which could used to solve the problem of bacterial diseases of tobacco in some degree.The results are showed as follows.1.In order to study the function of terminator(T19)in tobacco root-specific NtR19 gene.It was cloned in tobacco genome DNA by design of primers.The plant expression vector pBI121-GUSA-T19 including GUS gene was constructed,in which the terminator replaced the Tnos terminator in pBI121-GUSA vector.Tobaccos were transformed by Agrobacterium tumefaciens containing the constructed vectors.The transgenic plants were selected by PCR detection and GUS staining.The results showed that T19 could completely terminate gene expression by RT-PCR and the full-length mRNA 3 'UTR of NtR19 gene could be used as a terminator,which can solve the safety of exogenous gene in some extent and provides specific resource in tobacco genetic engineering.2.According to the expression vector p35S-loxP-GUS which was already constructed by the Cre/loxP system induced by self-deleted of antibiotics gene,four vectors were constructed through replacing the 35S promoter from self-deleted of antibiotics vectors,which connected different specific promoters(NtR12,NtR2,PP1),bacterial wilt resistance gene(Rip,Glu,Chi,NPR1)and terminator from tobacco(T19,Tp6)constructed to pSPROK vector.Vectors were respectively named:pLOXP-NtR12-Rip-T19,pLOXP-NtR12-Chi-T19,pLOXP-NtR2-NPR1-Tp6,pLOXP-PP1-Glu-Tp6.It will provide a guide for disease-resistant molecular breeding in tobacco.3.In order to obtain varieties resisting tobacco bacterial wilt,expression vector pLMAR-NtR2-NPRl-Tp6 was constructed according to the specific promoter NtR2,resistance gene NPR1 and terminator Tp6 which were already cloned in our laboratory.Tobaccos were transformed by Agrobacterium tumefaciens containing the constructed vectors.The transgenic plants were selected by PCR detection.The results showed that NtR2 promoter only expressed in tobacco roots.Bacterial wilt was inoculated in transgenic plants,the results also showed that Tp6 terminator could terminate the normal transcription of NRP1 gene and NPR1 gene could express which was induced from promoter.The transgenic plants had strong resistance to bacterial wilt.4.In order to study the function of NtR6 in tobacco,deletion promoter vectors were constructed according to the constructed expression vector pBI121GUSA-NtR6-1735 and promoter NtR6.The deletion vectors were named:pBI121GUSA-NtR6-1293;pBI121GUSA-NtR6-973;pBI121GUSA-NtR6-685;pBI121GUSA-NtR6-350.Tobaccos were transformed by Agrobacterium tumefaciens containing the constructed vectors.The transgenic plants were selected by PCR detection and GUS staining.The results showed that above-973bp fragments of NtR6 promoter could drive GUS gene express in roots,and-350bp?-685bp segment can drive the GUS expression in leaf and root not in stem,which showed in the segment of-685bp?-973bp the cis element had the function could inhibit the expression of leaf.While 0?-350bp segment had the cis element expression in the root and leaf.Above all,we could suggest that in the segment of-350bp-685bp had the enhancer element and preliminary revealed the function of NtR6 promoter.