关键词: 核盘菌, TRV-HIGS, 本氏烟, 分泌蛋白, 致病性, 实时荧光定量PCR

Abstract:

【Objective】 Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is the main problem in rapeseed planting in China, which causes serious yield and quality loss. Secretory proteins play an important role in the pathogenesis of pathogens. The genome of S. sclerotiorum contains a large number of genes encoding secretory proteins. The objective of this study is to identify and screen the secretory protein genes related to pathogenicity, reveal the pathogenic mechanism of S. sclerotiorum, and to provide an important target for the prevention and control of SSR. 【Method】 SMART software was used to analyze the protein domains of 8 candidate genes with signal peptides that were differentially expressed in the process of S. sclerotiorum infecting the susceptible and resistant Brassica oleracea lines, then the domains obtained by SMART analysis were annotated in SCOP, Pfam and PDB databases. The fragment with the length of around 300 bp in the encoding region of these genes was cloned into pTRV2 vector together with the GFP fragment. The suspension of pTRV1 was mixed equally with pTRV2-Gene and pTRV2-GFP, respectively. After 3 hours at room temperature, pTRV2-Gene vector and control (pTRV2-GFP) were transformed into 5-6 week-old leaves of Nicotiana benthamiana using syringe infiltration method. Subsequently, the infiltrated plants were cultured in dark for 48 hours and then grown in the normal light for 7 days. PDA mycelium blocks of S. sclerotiorum with a diameter of 6 mm were used to inoculate the infiltrated leaves of tobacco at the 9th day after transformation in vivo, in which the carrying surface was close to the leaves. After 48 hours of inoculation, the lesion size was measured and RNA from necrotic and infected tissues (around 1 cm from the edge of necrotic tissue) was extracted. qRT-PCR analysis was carried out to estimate the relative expression of target gene in N. benthamiana lines carrying TRV-HIGS vector. 【Result】 The putative functions of these 8 genes predicated with SMART and domain annotation were involved in the hydrolysis of proteins, nucleic acids or polysaccharides, the immunity response of host plants, and the tolerance to drugs and biotin synthesis of S. sclerotiorum. The average lesion area of control carrying TRV-GFP was 3.44 cm ² at 48 hours post inoculation of S. sclerotiorum. Except for one line (SS1G_07655), the lesion area of other 7 lines carrying TRV-HIGS vector was significantly lower than that of the control plants (P≤0.05), ranging from 1.63 to 2.61 cm ². qRT-PCR analysis showed that the gene expression level of these 7 genes in the TRV-HIGS lines was significantly lower than that of the control (P≤0.05). 【Conclusion】 Eight secretory protein genes with unknown function in S. sclerotiorum were successfully identified by TRV-HIGS technique. Seven genes related to the pathogenicity of S. sclerotiorum were screened out. Among them, SS1G_03146 with the strongest effect on the pathogenicity of S. sclerotiorum may be involved in the synthesis of biotin, and SS1G_04343 and SS1G_11912 may be involved in the immune response of host.

Key words: Sclerotinia sclerotiorum, TRV-HIGS, Nicotiana benthamiana, secretory protein, pathogenicity, qRT-PCR