Screening And Functional Analysis Of GSTF Genes Related To Sclerotinia Sclerotiorum Resistance In Brassica Napus

Brassica napus is a significant oilseed crop.Sclerotinia stem spot caused by Sclerotinia sclerotiorum is the most devastating disease in B.napus.This study was based on the results of previous studies of Glutathione-s-transferase(GST)gene family resistance in Arabidopsis thaliana,using RNAseq,RT-PCR techonology to screen pathogen or hormone-induced up-regulated genes from rape and obtain seven genes.According to the degree of induced expression,BnaC03g17120D,BnaA02g20640D,BnaC03g30870D and BnaA03g14140D were studied intensively.These four genes belong to the GSTF family.For the BnaC03g17120D,I mainly did some experiments to prove the result obtained by other graduated students in our lab.Based on the seedlings screened out in the previous period,the target protein was stably expressed in the transgenic plants and located on the cytoplasmic membrane by fluorescence microscope.This suggested that the positive seedlings were transgenic.The over-expressed plant was inoculated by S.sclerotiorum,and the inoculated leaves were subjected to ROS detection.The results showed that the average lesion size of the 35S::BnaC03g17120D-GFP overexpressed mutant leaves was 75.1 mm2,and the average wild type lesion size was 82.5 mm2.So the average lesion size of the overexpressed mutant was smaller than that of wild type.Trypan blue staining showed that the number of dead cells in overexpressed mutant leaves was less than that in the wild type leaves at 12 h and 24 h post inoculation,and NBT staining showed that the O2-accumulation level in the leaves of mutants was lower than that in wild type at the same time.All these results are the same as before and suggested this gene is important for plant resistant to S.sclerotiorum.In addition,we cloned BnaA03g14140D,BnaA02g20640D,BnaC03g30870D into different overexpression vectors(pEG103 or pCAMBIA3301)and transformed into the Nicotiana benthamiana by Agrobacterium-mediated injection of leaf tissue protocol.Then,the target genes were stably expressed in N.benthamiana leaves and S.sclerotiorum was inoculated in the injection area of the leaf.The inoculation results showed that the average lesion size of wild type was 160.6 mm2,while the average lesion size of 35S::BnaA03g14140D-GFP,35S::BnaA02g20640D-GFP and 35S::BnaC03g30870D-GFP were smaller than that of wild type.They were 83.4 mm2,144.9 mm2,and 104.7 mm2 seperately.These results preliminarily demonstrated that these four genes are likely to be closely related to the resistance to disease,pending further study.