Cloning And Functional Study Of BnGLIP1 In Brassica Napus L.

Brassica napus L.is the most important oil crop in China.It is widely planted in the middle and downstream of the Yangtze River.Not only can it be used to produce edible oil,but its meal are rich in protein and can also be used as animal feed.Sclerotinia sclerotiorum is an important plant pathogen worldwide,which seriously threatens the production of rapeseed in China.Chemical methods are often used to control S.sclerotiorum in production,but there are many problems.In recent years,the research on the function of plant GDSL lipase family genes has been gradually carried out,but there are few studies on the function of GDSL lipase in B.napus,especially in the field of anti-S.sclerotiorum.In this study,we screened a gene significantly associated with resistance to S.sclerotiorum in B.napus by candidate gene association analysis,and carried out a series of research work around this gene.The main results of this study are as follows:We cloned this gene,named BnGLIP1(B.napus GDSL LIPASE-LIKE 1),which is significantly related to S.sclerotiorum resistance in B.napus.Initially,we preliminarily understood the properties of BnGLIP1 protein through bioinformatics analysis.It belongs to plant SGNH lipase family.It consists of 360 amino acids and contains 20 kinds of amino acids.Its molecular weight is 39.5 KD.Its theoretical isoelectric point is 4.95.The predicted results show that the protein has a high degree of phosphorylation and may be a hydrophilic protein.This protein has signal peptide sequence,so we speculate that it may be exocrine protein.In order to better study the biological function of BnGLIP1,we detected the expression of BnGLIP1 in different tissues of B.napus by quantitative PCR.BnGLIP1 was expressed in all tissues,but the expression level was different,and the expression level was the highest in seeds.In addition,we also investigated the response of BnGLIP1 induced by biotic and abiotic stress in B.napus.It was found that S.sclerotiorum infection could induce up-regulation of BnGLIP1 expression.Exogenous salicylic acid,drought and high salt treatment can induce the expression of BnGLIP1.We constructed the recombinant expression vector pK7FWG2.0-BnGLIP1 and analyzed the subcellular localization of BnGLIP1 protein by tobacco transient expression system.The results showed that green fluorescence mainly appeared in the intercellular space of tobacco epidermis,suggesting that BnGLIP1 was expressed in the intercellular space of tobacco epidermis cells,which may be an exocrine protein.In order to study the biological function of BnGLIP1 in the plant antibacterial nucleus disease,we used the tobacco transient expression system to transiently express BnGLIP1 in tobacco,and found that the gene enhanced the resistance of tobacco to S.sclerotiorum.We further overexpressed the BnGLIP1 gene in Arabidopsis,and found that it enhanced Arabidopsis resistance to S.sclerotiorum.BnGLIP1 overexpression lines and interference lines were established by genetic transformation system of Hypocotyl in B.napus.On the one hand,this study will help us understand the molecular mechanism of plant response to S.sclerotiorum infection,on the other hand,it will provide candidate genes for molecular improvement of rapeseed varieties and breeding of new disease-resistant germplasms in the future.