Analysis Of The Monoacylglycerol Lipase Gene Family In Brassica Napus And Characterization Of The BnA9::BnaC.MAGL8.b Male Sterile Plants

Oilseed rape is an important oil crop in our country and the world and it is vital to study lipid-related genes in this crop.Lipids,as one of the three nutrients for living creatures,affect many physilogical processes.Lipid anabolism and catabolismprocess,among those basic biological processes,is wildspread in many species.The homolog genes show conserved biological function in different species.In animal,monoacylglycerol lipase(MAGL)participates in fat mobilization and assimilation,and it also affacts many physilogical processes by regulating endocannabinoid signalling.In plants,there are few reports on this gene family.As far as we know,there is only one paper characterized the molecular and biochemical properties of MAGL family in Arabidopsis(AtMAGL).To increase our knowldge on lipid metabolism related genes and discovery new valuable genes,we characterized MAGL gene family in Brassica napus(BnMAGL)and collected basic information on 45 potential BnMAGL genes.Furthermore,we functional characterized several genes with the transgenic plants in Arabidopsis.The main resutls are:(1)In this study,we found 45 BnMAGL genes based on the AtMAGL protein sequences.And they were classified into 7 groups by their protein sequence similarity.These BnMAGLs show similar gene structure as AtMAGLs.(2)The expression patterns of these BnMAGL genes were characterized with transcriptome data from GEO and Quantitative Real-time PCR.Most of them are preferentially expressed in developing seeds and are induced by Sclerotinia sclerotiorum and Leptosphaeria maculans inoculation.In addition,IAA treatment does not affect their expression.(3)In vitro enzyme assays were conducted with six BnMAGL recombinant proteins,and five showed MAGL activity.The potential functions of 15 BnMAGLs were studied on the transgenic plants in Arabidopsis.The seed oil content was decreased in 10 transgenic plants.The seed oil content was same in the 35S::BnaC.MAGL8.b transgenic plants and wild type.The 35S::BnaC.MAGL8.b seeds contained more oleic acid,and the molar percent of palmitic acid and linolenic acid were decreased in the 35S::BnaC.MAGL8.b seeds.(4)Semi-thin sections,transmission electron microscopy,scanning electron microscopy,and transcriptome analysis were employed to decipher the molecular mechanism underlines the male sterile BnA9::BnaC.MAGL8.b plants.Alexander's stanining showed the BnA9::BnaC.MAGL8.b anthers contained two types of pollen grains: the round pollen grains and the flat pollen grains.The flat pollen grains were defetive.These two types of pollen grains were attached with each other and stuck to the locue.According the histology results,the tapetal cells became vacuolated at stage 8 and the vacuolation became severe at stage 10.Finnally,the tapetal cells degenerated at stage 12.Most microspores were degraded around stage 10,and only few round pollen grains presented in the locule at stange 13.The transmission electron micrographs indicated a defected pollen wall in round pollen grains from the transgenic plants,characterized with the irregular baculae structure and limited pollen coat.Transcriptome data and Quantitative Real-time PCR analysis indicated AMS and its downstream genes(related with pollen wall formation)were down-regulated in the transgenic plants;on the other hand,jasmonate signalling related genes and reactive oxygen species homeostasis related genes were up-regulated.Based on the histology results and gene expression data,we proposed a molecular model for the male sterility in the BnA9::BnaC.MAGL8.b plants.