Analysis And Cloning Of Sclerotinia Resistance Gene Qa-SNARE Family In Brassica Napus

Oilseed rape is one of very important oil crops in our country.Sclerotinia stem rot caused by Sclerotinia sclerotiorum,witch is a fungal disease,poses a great threat to rape production.This disease cause more than 80%yield losses,when the infected rape plants yield decreased by 10%to 70%.At present,the traditional agricultural control and chemical control are still the main control method for Sclerotinia sclerotiorum.As effective control measures;the highly resistant materials of S.sclerotiorum are still not cultivated.SNARE complex,short of soluble N-ethylmaleimide-sensitive factor attachment protein receptors,plays an important role in plant physiological metabolism and plant defense responses against pathogens.The research of SNARE gene family in rape was poor,while in the model plant Arabidopsis is deep.There are nine SYP1 family genes,which belong to Qa-SNARE family,located in the plasma membrane.To study the function of SYP1 syntaxin in Brassica napus further,we use the nine SYP1 groups of Arabidopsis syntaxins as probes to search for their homologous genes in B.napus database.In this research,physical and chemical properties,subcellular location,conserved domains,secondary and tertiary structure of protein,and phylogenetic tree were predicted and analyzed by the bioinformatics method.The results showed that there were 27 SYP1 syntaxin related proteins,located in cytoplasm,nucleus,endoplasmic reticulum,and vesicle secretion system,respectively.And they all belong to fusion protein superfamily.There is a high similarity among these 27 sequences,as well as a high homology with Arabidopsis SYP1 syntaxin family,The phylogenetic tree indicated that B.napus SYP1 syntaxin and Arabidopsis SYP1 may originate from a common ancestor.In order to explore new gene resource having the resistance to S.sclerotiorum of rape,further analyze the interaction mechanism between the rape and S.sclerotiorum,we first performed the temporal and spatial expression analysis of 8 BnSYP1 genes of rape.Then in vivo inoculation of two different varieties of oilseed rape was performed,further real-time fluorescence quantitative PCR was used to analyze expression of every gene at the time interval 6h and 12h.The results showed that the gene BnaA04g05470D was up-regulate significantly,indicating the gene plays an important role in interaction between the rape and S.sclerotiorum.To further determine the function of BnaA04g05470D in the interaction,two over expression vectors PEG 103-BnaA 04g05470D and PEG 104-BnaA04g05470D were constructed and successfully transferred to Arabidopsis wild type Col-0 through the method of floral dip transformation.We screened for T1 generation with herbicide Basta,laying the foundation for the deeper research on specific gene function in the process of rape resistant to S.sclerotiorum.