| Both ginsenosides and phytosterols can be biosynthesized through the mevalonate pathway and have the same precursor material,2,3-oxidosqualene.There are five metabolic branches of 2,3-oxosqualene in the mevalonate pathway of ginseng,of which two branches are related to the synthesis of ginsenosides,two branches are related to the synthesis of phytosterols,and the other is related to the synthesis of lupeol.Lecithin sterol acyltransferase(LCAT)can acylate phytosterols to sterol esters.Silencing this gene may result in the accumulation of free sterols in plants,which may feedback inhibits two metabolic branches in the downstream of 2,3-oxosqualene that are not related to the synthesis of ginsenosides,and then makes more 2,3-oxosqualene flowing to ginsenoside synthesis branch.Study on the function of lecithin sterol acyltransferase gene in the synthesis of ginsenosides is beneficial to regulate the content of monomeric saponins and total saponins of ginseng from the perspective of metabolic.In this study,the 248,993 unigenes database and the expression quantity database of laboratory were used,through the bioinformatics analysis of the LCAT gene family in ginseng,7 genes were screened and analyzed.By analyzing the expression of the 7 genes in ginseng hairy roots which induced by MeJA,PgLCAT5 was chose for gene cloning and overexpression vector constructing,and the Agrobacterium-mediated transformation of carrots was performed to further verify the function of the gene,it will lay a foundation for the research and utilization of important functional genes in ginseng in the future.The results showed that:1.The blast and annotation of 7 PgLCAT genes showed that the length of them were between 400 bp and 2000 bp,The homology comparisons of the sequences showed that the most homologous species were Daucus carota subsp.sativus and Aegilops tauschii subsp.tauschii,which indicating that the ginseng LCAT gene may closely related to them in origin and evolution;2.The phylogenetic tree showed that 7 PgLCATs were divided into three branches,3 were closely related to yellow carrots,the other 3 were closer to the genetic distance of Aegilops tauschii,and the last one was closer to the genetic distances of tobacco,tomato and potato,which in line with previous results;3.The results of expression patterns showed that in 14 ginseng tissues,PgLCAT1,PgLCAT2 and PgLCAT7 were actively expressed in the stalk,and PgLCAT5 was actively expressed in the leaves.In 42 cultivars,PgLCAT1 had specificity regional expression in the S29 farm cultivar.4.The interaction network of 7 PgLCAT and ginsenoside synthesis key enzyme genes showed that PgLCAT5 was highly related to the key enzyme genes.Therefore,the PgLCAT5 gene was involved in the biosynthesis of ginsenosides obtained from the above results of bioinformatics analysis.5.The correlation analysis between the expression of PgLCATs and ginsenoside content showed that there was a significant positive correlation between the expression of PgLCAT5 and the contents of ginsenoside Rb1 and total saponins,which were consistent with the results of real-time quantitative PCR.6.In ginseng hairy roots induced by MeJA elicitor,some ginsenoside saponin content and total saponin content were significantly changed.Moreover,the expression pattern trend of PgLCAT5 gene and some ginsenoside synthesis key enzyme genes,Dammarenediol synthase(DS),squalene epoxide(SE)and cytochrome P450(CYP450)were consistent.Therefore,it is speculated that the PgLCAT5 gene was involved in the biosynthesis of ginsenosides obtained from result of elicitor regulation.7.In this experiment,the PgLCAT5 gene was cloned and the 1438 bp full-length gene fragment was obtained.The pBI121-PgLCAT5 overexpression vector was successfully constructed.A total of 10,394 carrot explants were transformed to obtain 111 resistant seedlings.After PCR and hybridization detection,37 of them were positive transgenic plants,and the conversion rate was about 33.33%. |
