Studies on improvement of Chinese wheat cultivars for resistance against an endemic virus using transgenesis and virus-wheat interactions at the molecular and ultrastructural levels
The present study was aimed at the improvement of Chinese wheat cultivars for resistance to Soil-borne wheat mosaic virus (SBWMV) by transgenesis, and an examination of virus movement by studying the complementation of movement proteins (MPs) from Tobacco mosaic virus (TMV) and SBWMV using a TMV-based virus vector. A number of SBWMV coding sequences, including the functional coat protein (CP) gene, dysfunctional MP gene, and functional and dysfunctional replicase complex sequence, were cloned into an expression vector pUbi35S and the expression of those viral genes under the control of maize ubiquitin-1promoter was tested in transfected tobacco protoplasts. Parameters for the bombardment of immature embryos using the biolistic gun were optimised through transient expression of a chimeric pglucuronidase (uidA) gene. Transgenic wheat for the model variety Bob White and three Chinese semi-winter wheat cultivars were obtained by microprojectile bombardment of pre-cultured immature embryos using achimeric bar gene as the selectable marker. Plants were regenerated under phosphinothricin (PPT) selection. The transgenic nature of the regenerated wheat plants was demonstrated by Southern hybridization analysis. Expression of viral CP gene was confirmed from most CP-transgenic wheat by RT-PCR testing, while only one CP-transgenic plant produced CP at a detectable level. The meiotic stable transmission of transgenes in several transgenic lines was confirmed by polymerase chain reaction (PCR) analysis of R1 progenies. ELISA testing of several selected transgenic lines mechanically inoculated with Chinese wheat mosaic virus (CWMV), a new member of Furovirus genus, revealed that most of the lines were infected by the virus, while some plants from several lines appeared not to be infected. A new selection approach based on the phosphomannose isomerase (pmi) gene as the selectable marker, and mannose as the selective agent,was developed to produce transgenic wheat plants for both Bob White and Chinese wheat varieties. The introduction of SBWMV CP gene into Chinese wheat cultivars was accomplished using the mannose selection system, and Southern analysis confirmed the integration of transgenes in the genome of transgenic wheat. An expression vector was constructed by cloning the putative SBWMV MP coding sequence into a TMV-based vector in which the native TMV MP gene was rendered defective by open reading frame shift. When using this chimeric virus to infect tobacco plants, it was found that the 37 kDa protein (p37) encoded by SBWMV could be functionally complementary to the 30kDa of TMV MP, this also confirmed that the p37 is the MP of SBWMV.
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