Study On Protein Analysis Of Liquid Exudate Of The Sclerotinia Sclerotiorum(Lib.)De Bary And Ocimene-induced The Resistance To Sclerotinia Sclerotiorum Of Brassica Napus

Sclerotinia stem rot is a serious fungal disease caused by Sclerotinia sclerotiorum (Lib.) de Bary which affects the growth of oilseed rape and the yield and quality of oilseed rape. sclerotia formation is an important process in the cycle of infection. In order to study the proteins from the liquid exudate of developement sclerotia which function in infection. In this study, the liquid exudate of developing Sclerotinia sclerotium separated by SDS-PAGE. High abundance of seven protein bands were isolated. After mass spectrometry, eight kinds of proteins was determined and the gene cloned, prokaryotic expression and functional analysis of protein have done. At the same time, some reports shown that ocimene can improve plant disease resistance. Treated Brassica napus with ocimene to study ocimene whether can enhance disease resistance of Brassica napus and the possible mechanism of resistance, the main results are as follows:1. Protein bands which separate from the liquid exudate were analyzed by mass spectrometry. According to the results of mass spectrometry and the protein sequences blast in Mascot database, eight protein from liquid exudate of developing Sclerotinia sclerotium was determined. Using the reference nucleotide sequences on the NCBI web site to design primers.7 full-length cDNA sequences of Hyp-2?Hyp-3?Man-41?Hyp-42?Amy-5?SSL-6 and Hyp-7 were cloned from developing sclerotia by RT-PCR technology. Addition 217 bp in cloned Hyp-3 gene which compared with the NCBI website sequences, Amy-5 clones of exon length was 1500 bp.2. Currently successfully constructed pGEX-4T-1-Hyp-2, pGEX-4T-1-Hyp-3, pGEX-4T-1-Man-41, pGEX-4T-1-Hyp-42, pGEX-4T-1-SSL-6 and pGEX-4T-1-Hyp-7 total 6 prokaryotic expression vector.3. Above 6 prokaryotic expression vector in E.coli BL21(DE3)pLysS strain induced with IPTG to express recombination protein. Finally the Hyp-3, Man-41, Hyp-42 and SSL-6 a total of four protein expressed successfully. Hyp-42 protein expressed as inclusion body, tried to crack inclusion bodies, refold by dialysis and purify, but did not succeed. Hyp-3, Man-41 and SSL-6 protein were soluble protein partly and then purified by GST column chromatography. Hyp-3 was purified unsuccessfully, Man-41 and SSL-6 protein were purified successfully.4. After bioinformatic analysis, Man-41 protein have conical pocket structure, signal peptide and combined with calcium ions at the Thr514. Man-41 protein domain belong to glycoside hydrolase family 47 and the a-mannosidase essential catalytic active sites:Glu125, Arg129, Asp270, Ser271, Glu274, Arg420, Glu422, Glu425, Glu485, Thr514 and Glu515 also in Man-41 protein sequence. Purified Man-41 protein react with specific mannosidase substrate PNPG, the a-mannosidase activity was 27.67 U/mg. Adding calcium ions in reaction system, there was no significant differences in enzyme activity. Purified Man-41 protein detected by ICP-MS, the result show that the protein has been bind calcium ions.5.After bioinformatic analysis, SSL-6 protein is fungal lectin and have typical AFL1 six-leaf fan structure. At present, pCAMBIA1303-SSL-6 overexpression vector (GUS, GFP reporter gene) has successfully constructed. Using Arabidopsis thaliana as object to study SSL-6 protein function, SSL-6 gene have been transfromed into Arabidopsis thaliana. Ti generation of transgenic Arabidopsis seeding has obtained and SSL-6 gene express in Arabidopsis at RNA and protein level detected by RT-PCR and GUS technique and lay the foundation for subsequent experiments of the resistance to Sclerotinia stem rot.6.XY-15(resistance) and 98C40(susceptible) Brssica napus treat with ocimene 24h. In XY-15 GLU, NPR1, VSP, AOS, GST, PGP, OXO and CHS can be induced significantly (two fold expression) by ocimene. In 98C40, PR1, CHI, ICS1, NPRI, LOX2, PDF1.2, AOS, MYC2, GST, PALI and PGIP can be induced significantly. Two kinds of materials are compared, the susceptible materials 98C40 induced resistant genes significantly than the resistant materials XY-15 three genes, but the XY-15 material can induced GLU, VSP, AOS, OXO and CHS gene significantly, while 98C40 was not significantly.7. Brassica napus treat with different concentration ocimene and inoculated with Sclerotinia sclerotiorum 4 days. In XY-15, NPRI, AOS, MYC2, GPX1 and PALI express significantly, in 98C40, GLU, PDF 1.2, MYC2, PGIP, PAL1 and OXO express significantly. The number of gene express significantly in susceptible materials 98C40 was more than resistant materials XY-15 one gene. But the resistant materials can expression significantly of NPR1, AOS, GPX1 while the expression of 98C40 was not significantly.