Research On Development And Application Of Artificial Promoter Based On TALE

Bacterial blight (BB) and bacterial leaf streak(BLS) of rice,caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv.oryzicola(Xoc), respetively, are the representative diseases resulted in the most severe reduction of rice output. The cultivation of resistant varieties was considered asthe most effective and economical option to control diseases. Up to now, none of gene with a broad spectrum resistance to BB and BLS was observed in rice. When it had already been discovered that transcription activator-like effectors (TALE) from Xanthomonas pathogens act as transcription factors in the host plant, deciphering the modular code of DNA binding specificity of TAL effectors.Which offered an opportunity to create resistant plants using the artificial promoter induced by Xoo and Xoc based on TALE.In this study, we planed to breeding resistance rice with broad-spectrum to BB and BLS using inducible artificial expression promoter by Xoo and Xoc, which will provide a novel clue and method for functional analysis of resistance gene and breeding of resistant rice, and afford gene resources for rice breeding of resistant rice to Xoo and Xoc.In order to identify activity of artificial promoter based on TALE in rice, we firstly constructed an expression vector using inducible promoter to drive GUS reporter gene, consequently, we builded a serial of constructs to express different functional fragment of Xa21, a resistance gene to BB, in rice. These constructs were introduced into Agrobacterium tumefaciens LBA4404by electroporation, and subsequently transformed toThe main results are as follows:1. The plant expression vectors XooTaleP-1301, XooTaleP-Xa21, XooTaleP-Xa21pk, XooXocTaleP-1301, XooXocTaleP-Xa21, XooXocTaleP-Xa21pk and pCAMBIA1301were successfully constucted using for rice transformation.2. Seven binary expression vectors were transferred into TP309rice callus, respectively, through agrobacterium-mediated transformation system. After selection, differentiation, rooting, transgenic lines of total seven vectors were obtained. The transgenic plantlets were then transplanted in paddy field. The results showed that all transgenic lines were positive after testing the To transgenic lines by PCR. The number of positive transgenic plants were24,10,15,46,88,35, respectively.3. After treat transgenic rice XooXocTaleP-1301with rice bacterial leaf blight pathogen PXO99and rice bacterial streak virus RSI05for2days, we use GUS qualitative and quantitative analysis the transgenic lines XooXocTaleP-1301, in order to find out whether the expression of XooXocTaleP was induced by rice bacterial leaf blight pathogen and rice bacterial streak virus. The result shows that the expression of XooXocTaleP was induced by rice bacterial leaf blight pathogen and rice bacterial streak virus; After treat transgenic rice XooTaleP-1301with rice bacterial leaf blight pathogen PXO99processing for2days, we use GUS qualitative and quantitative analysis the transgenic lines XooTaleP-1301, in order to find out whether the expression of XooTaleP was induced by rice bacterial leaf blight pathogen, the result shows that the expression of XooTaleP wasn’t induced by rice bacterial leaf blight pathogen.4. Two days after inoculating rice bacterial leaf blight pathogen PXO99to XooXocTaleP-Xa21, XooXocTaleP-Xa21pk and pCAMBIA1301, sampling the leaf at the snip part. Respectively random extract eight different strains from transgenic of XooXocTaleP-Xa21and XooXocTaleP-Xa21pk, three different strains from transgenic of pCAMBIA1301and one strain from transgenic of CX8621and CX62121B. With semi-quantitative RT-PCR detection technology test whether exogenous gene of XooXocTaleP-Xa21and XooXocTaleP-Xa21pk, Xa21, was expressed in transgenic plants in RNA level (transgenic of pCAMBIA1301, CX8621and CX6221B was negative control). The Semi-quantitative RT-PCR results show that the exogenous gene of XooXocTaleP-Xa21, Xa21, has expressed at the RNA level, and the expression of Xa21is higher than the control group in three plants, the expression of the rest were equal with the control group; the exogenous gene of XooXocTaleP-Xa21pk, Xa21, has expressed at the RNA level too, in one plant the expression of Xa21is higher than the control group, the expression of Xa21is lower than the control group in two groups, the expression of the rest were equal with the control group..5. After inoculating rice bacterial leaf blight pathogen PXO99to XooTaleP-Xa21, XooTaleP-Xa21pk, XooXocTaleP-Xa21, XooXocTaleP-Xa21pk and pCAMBIA1301for half a month. The results show that the lesion length of part of transgenic lines of XooXocTaleP-Xa21, XooXocTaleP-Xa21pk are shorter than the control group which is transgenic lines of pCAMBIA1301, which indicate that they had a certain degree of resistance. While the transgenic lines of XooTaleP-Xa21, XooTaleP-Xa21pk didn’t have the obvious disease resistance.