Identification Of RNA Interfering Fragments Against Fusarium Head Blight Disease And The Mechanism Of A Maize VP1 Gene In Regulation Of Pre-harvest Sprout In Wheat

Fusarium head blight(FHB)in wheat is a devastating disease in China,not only causing huge losses of yield but also producing mycotoxins.By far,FHB has not been well controlled and the main reasons for that are deficient of resistance germplasm against FHB.Pre-harvest sprout(PHS)of wheat is an important disaster in main wheat production areas in China,resulting in severe reduction of yield,quality and seed viability.Introduction of a maize Vp1 gene into wheat genome enhanced PHS tolerance.However,the mechanism of the maize Vp1 gene in regulation of PHS in wheat is not well known.In this study,two parts of work have been carried out.In the first part,four RNAi fragments have been used for in vitro transcription to produce double-strand RNA(ds RNA),and all the four ds RNAs showed an effective inhibitory effect on growth of FHB pathogen Fusarium graminearum in vitro.These RNAi fragments and other RNAi also derived from F.graminearum have been constructed as plant expression vectors together with lemma-specific promoter Lem2 or constitutive promoter Ubiquitin.The constructed vectors were transferred into wheat cultivars via Agrobacterium-mediated transformation.A total of 25 transgenic wheat lines were generated and inoculated with FHB pathogens.The obtained main results from FHB resistance evaluation are as follows:(1)For transgenic wheat lines transform with RNAi Chs7-3-Chs7-4 regulated by Lem2 promoter,inoculation of T5 generation at flower stages with FHB pathogen revealed that two lines(YULC2,YCLC)displayed 37%-43% significantly higher resistance than the non-transgenic control Yangmai 158.As for T6 generation,three transgenic lines(YULC2,YULC4,YCLC)had 37%-49% significantly higher resistance compared with that of the control Yangmai 158;moreover,the harvested grains of these three lines had remarkably reduced mycotoxins,with a reduction ranging from 53%-92% relative to that of control Yangmai 158.(2)For transgenic wheat lines transform with RNAi Chs7-3-Chs7-4 regulated by Ubiquitin promoter,inoculation of T3 generation at flower stages with FHB pathogens in greenhouse showed that two lines(YU1,YU6)had 38%-39% significantly enhanced resistance compared with that of the non-transgenic control Yangmai 158.Furthermore,inoculation of T3 generation at seedling stages revealed that five lines(YU1,YU3,YU4,YU6,YU7)had very significantly enhanced resistance,ranging from 17%-36%,compared with that of the control Yangmai 158.(3)For transgenic wheat lines transformed with RNAi fragments from five different genes,Chs3b-5-Pkc-5-Chs7-4-Gls-6-Myo II-14,under control of Ubiquitin promoter,inoculation of T3 generation at flower stages with FHB pathogens in greenhouse revealed that one line(5C3)had 24% higher resistance than the non-transgenic control Fielder.At seedling stages after inoculation,two lines(5C1,5C3)showed 17%-23% very significantly higher resistance than the control Fielder.Furthermore,one line(5CY1)had 77% very significantly higher resistance than the non-transgenic control Yangmai 158 in T3 generation after single-floret injection in greenhouse,while one line(5CY2)had 62% significantly higher resistance than the control Yangmai 158.In the second part of work,comparatively study of transcriptomes and proteomics between a Vp1-transgenic wheat line and its control without the Vp1 gene was performed.The results showed that during wheat seed development Vp1 regulated transcript levels of 0.7%-7.7% genes,whereas the contents of 1.1%-3.4% proteins were changed including storage proteins,hydrolysis proteins and dehydrin etc.Combined with the metabolic pathways based on transcriptomes and proteomics data,we found that the maize Vp1 gene appears to regulate the key genes involved in signal transduction pathways of hormones and to participate in the interaction between abscisic acid and other plant hormones to finally enhance seed dormancy and PHS in wheat.In this study the RNAi fragments and transgenic wheat lines obtained provide materials and evidences for wheat resistance improvement of FHB;the mechanism of pre-harvest sprout mediamediated by the VP1 gene revealed supplies information and support for the utilization of a maize Vp1 gene in enhancement of PHS in wheat.