| The fungal pathogen Sclerotinia sclerotiorum (Lib) de Bary causes adestructive disease, one of the main diseases in oilseed rape (Brassica napus).Use of resistance varieties is economic and effective measures for control of thedisease. However, variesties with high resistance are at present lack, themolecular mechanisms are beyond understanding and there is a demand fordevelopment and application of innovative technologies to discover keyresistance genes for resistance improvement. In the time that great researchprogress in plant functional genomics and gene engineering have rapidly beenmaking, particularly in molecular network of resistance in the model plantArabidopsis thaliana, it is significant that, by transfer or development of theinnovative technologies, cloning and investigation of functional genes forresistance improvement of oilseed rape.In this study, we first detected gene expression induced by different diseaseresponse signaling molecules to look at whether disease response pathwaysfound in A. thaliana exist in B. napus. Based on this,three genes were selectedwith putative function for disease resistance and transformed into oilseed rape inorder to elucidate experimentally gene functions and their value for breeding.In induced resistance experiments, chemical inducers used to spray leavesare defense response signaling molecules benzothiadiazole-7-carbothioic acid(BTH, SA analog) and methyl jasmonate (MeJA), a toxin oxalic acid (OA)produced by S. sclerotiorum during its pathogenesis. After treatments, theexpression of seven marker genes in defense signaling pathways includingPDF1.2 (JA-dependent signaling pathway), PR-1 (SA-dependent pathway),NPR1(modulating above pathways), APX(associated with the oxidative burstpathway) and other three pathogenesis-related protein genes (PR-5, GLU andCHI) was detected by semi-quantified RT-PCR. The results showed that thesechemical treatments and the pathogen inoculation can induce up-regulatedexpression of all these genes although their degree are various, suggesting thethree important signaling pathways could exist in oilseed rape.In gene function investigation by gene transformation, first, bioinformaticanalysis was conducted based on a UniGene library constructed from leavestreated by above chemicals and inoculation with S. sclerotiorum. Based on theinduced experiments and the bioinformatic analysis, genes with putativefunctions in resistance were selected for vector construction and genetictransformation. Three genes selected were named as BNT19K4,BNF7A10 andBNF5011, respectively, all are with full length sequences including all ORFs.Plant overexpression vectors,named as pGTK,pGFA and pGFO,respectively, were constructed based on a starter vector pG4A. Two RNAinterference vectors, pGRTK and pGRFO, were constructed based on a startervetor pG4A-intron. All of these vectors include a gene bar conferring resistanceto herbicide glufosinate-ammonium (basta). The genetic transformationmethod is an innovative methods newly developed in our lab (like the pollentube-mediated one). In total, 13002 and 14701 transformed seeds foroverexpression and RNAi, respectively, were harvested. After screening withherbicide, there are 305 plants resistant to the herbicide (RNAi has not beenscreened). In PCR identification, there are about 80% positive plants. Intransformation into B. rapa, there are 94 seedlings resistant to herbicide and thesame rate as that in B. napus. And in the same, about 80% is PCR-positive.Detached leaf inoculation test were undertaken in order to assess theresistance of transgenic plants to S. sclerotiorum. These plants transformed withoverexpression vectors, pGTK,pGFA and pGFO, respectively, werePCR-positive. The results showed that transgenic plants increased in resistancelevels in comparison with the control. Analysis indicated that infection andlesion development were delayed, indicating that transgenic plants enhancedresistance to S. sclerotiorum.
|
PDF Full Text Request