Screening And Characterization Of The Encoding Genes For Protein Partners From Brassica Napus And Stellaria Apetala Ucria That Interacted With Pg, A Virulence Factor From Sclerotinia Sclerotiorum

Oilseed rape(Brassica napus) is an important industrial crop and a major oil crop.It plays important roles in national economy and agricultural production.Sclerotinia stem rot caused by Sclerotinia sclerotiorum is the most devastating disease of oilseed rape in China. To date,no complete resistance has been found in rapeseed germlpasm and its relatives.It is of very significance to understand the mechanisms from molecular level of the interactions between Brassica napus and Sclerotinia sclerotiorum and to isolate genes associated with susceptibility and resistance to the infection by Sclerotinia sclerotiorum, which should be helpful to protect plant from infection.In this paper,yeast two hybrid (Y2H) was used to screen the PG-interacting protein factors.We have succeeded in constructing the cDNA libraries of RS-1,a rapeseed breeding line with partial resistance to Sclerotinia sclerotiorum and of Stellaria apetala Ucria,a weed in rapeseed field that is immune to Sclerotinia sclerotiorum and also screened some genes using PG as bait which is regared as an important effector of Sclerotinia sclerotiorum.A C2-domain gene was isolated in rapeseed cDNA library by Y2H with PG as bait.Its expression pattern in different organs was analysed by semi-quantitive PCR and Real Time PCR.The subcelluler localization of the C2 protein was performed by transient expression in epidermal onion cells.A transmembrane protein and some other proteins assumed to be involved in stress responses were isolated in Stellaria apetala Ucria cDNA library by Y2H with PG as bait. The main results are as follows:1.Two primers was designed based on the conserved sequences flanking ORF of PG genes published in NCBI(accession numbers L29040,L29041,L12023, AF501307,AF501308,and AY312510),sspg1d(NCBI accession number: AF501307),one of PG genes which is highly expressed and mostly related to pathogenecity in Sclerotinia sclerotiorum was clone by RT-PCR,it was subcloned into the pGBKT7 vector and used for screening ofcDNA library.2.Two cDNA libraries of Brassica napus and Stellaria apetala Ucria induced by Sclerotinia sclerotiorum were constructed,and some candidate genes postulated to be related to Sclerotinia stem rot were isolated by screening the cDNA libraries. Two interesting genes assumed to be involved in PG signaling named IPG-1 and XFL-PG-1 were further analyzed respectively.3.The expression of IPG-1 was analyzed by semiquantitative RT-PCR and real time PCR,the result showed that its expression was greatly induced in leaves and relatively low in stems and floral organs when the rapeseed plant was inoculated with Sclerotinia sclerotiorum;The yeast two hybrid assay showed that the C terminal domain of IPG-1 protein contributed to the interaction with PG.The transient expression of IPG-1 in epidermal onion cells indicated that IPG-1 was maily located in cytoplasm while the protein of XFL-PG-1 is located on plasma membrane.4.The ORF of IPG-1 was cloned into pJIT166-GFP,pBINPLUS vectors to construct the over-expression vector,and a partial sequence of the IPG-1 was inserted into the RNAi vector to study the function of IPG-1;In order to test the interaction between IPG-1 and PG in vitro,the ORF of the two genes was cloned into pET30a and pGEX4T respectively.Homolog of IPG-1 in Arabidopsis thaliana was cloned and its RNAi constructs was constructed.PAC1,a transcription factor involved in PG production in ScIerotinia scIerotiorum was cloned and its RNAi constructs was constructed for RNA interfere analysis.