Screening And Cloning Of Candidate Genes For The Biosynthesis Of Petroselinic Acid In Coriandrum Sativum L. Fruits

Petroselinic acid is a monounsaturated ?-6 fatty acid,which is an isomer of oleic acid due to their different double bond position.And this specific characteristic results in quite different properties of the two fatty acids.Petroselinic acid shows great prospect in industrial application and human nutrition.Coriandrum sativum L.,belonging to umbelliferae,contains a large content of petroselinic acid in fruits(up to 80%).Therefore,the fruits are considered to be an ideal material to investigate the biosynthesis mechinism of petroselinic acid.However,due to the limitation of methods and techniques,no great progress has been made in recent 20 years on the biosynthesis mechinism of petroselinic acid.Thanks to the widespread usage of next generation sequence,RNA-seq shows great advantages in illustrating molecular mechinism of development and metabolism.In this research,accumulation pattern of petroselinic acid in Coriandrum sativum L.fruit was determined for guiding RNA-seq sampling.Candidate genes for the biosynthesis of petroselinic acid were screened using RNA-seq of fruits from different developing stages.The possible molecular mechanism of petroselinc acid biosynthesis in Coriandrum sativum L.fruit was preliminarily investigated.The amount of candidate genes was redeced by gene tissue-specificity analysis and the differentially expression patterns.Based on the sequences obtained from RNA-seq,five candidate genes which are most likely to participate in biosynthesis of petroselinic acid were cloned and their seed-specific expression vectors were constructed respectively.The main results obtained in this research are summarized as follows:(1)The fatty acid profiles of Coriandrum sativum L.fruits from seven development stages were analyzed.Both relative contents of petroselinic acid in the total fatty acids and in the dry weight showed “S” curve.Based on this analysis,the rapid accumulation period of petroselinc acid was determined,which is from DAF9 to DAF21.(2)Six samples from 3 stages in this rapid accmulation period were sent for RNA sequencing and 96,350 Unigenes and 114,513 transcripts were obtained.Further function annotation and transcriptional analysis were also carried out.Combining the dynamic accumulation profile of petroselinic acid in fruits and the biosynthesis mechinism of fatty acids,sixteen candidate genes were sreened,including three genes of acyl-acyl carrier protein desaturase(ACPD),three genes of 3-ketoacyl-acyl carrier protein synthase(KAS),one gene of acyl-acyl carrier protein thioesterase B(FatB),one gene of 2-diacylglycerol acyltransferase(DGAT2),two genes of phospholipid:diacylglycerol acyltransferase 1(PDAT1),one gene of Glycerol-3-phosphate acyltransferase(GPAT9),three genes of lysophosphatidic acid acyltransferase(LPAAT)and two genes of ysophosphatidylcholine acyltransferase(LPCAT).And the transcriptional levels of some candidate genes were further determinded by real-time PCR.And the possible molecular mechanism of petroselinc acid biosynthesis in Coriandrum sativum L.fruit was preliminarily discussed.(3)The expression pattarns of ACPD,KAS,FatB,DGAT2 and other family genes in Coriandrum sativum L.fruit and leave were detected by semi quanatitative RT-PCR and real-time PCR,which provided information to reduce the amounts of candidate genes involved in the biosynthesis of petroselinic acid.Among these genes,CsACPD1,CsACPD4,CsKAS I-2,CsFatB and CsDGAT2 were cloned and seed-specific expression vectors of these five genes were constructed respectively.