Discovery Of Capsicum Acyl-CoA Synthase Gene And Its Relationship With Capsaicin Synthesis

Capsaicin is the substance basis of pungency in pepper,the content of capsaicin determines whether the pungency is high or low directly.The pathway of capsaicin synthesis has been explained at the chemical level,while the key enzymes in other steps and the genes responsible for coding these enzymes have not been fully functional identified except capsaicin synthase.The predecessors mostly used Annual peppers(Capsicum annuum L.)as materials to study the mechanism of capsaicin biosynthesis,but few reports used Chinese peppers(Capsicum Chinense Jacq.)with high pungency.It has been proved that the last step of capsaicin synthesis is catalyzed by capsaicin synthase(CS).The two precursors of capsaicin synthase are catalyzed by phenylalanine pathway and branched-chain fatty acid pathway,respectively.Previous studies have concluded that the last step of the branched-chain fatty acid pathway is catalyzed by acylcoenzyme A synthase(ACS),but the gene encoding the enzyme has not yet been identified.In this study,Hainan yellow lantern and Bhut jolokia in C.chinense were taken as research objects.First,The content of capsaicin in pepper fruits at different developmental stages and tissues was determined.Then,the ACS genes in pepper was systematically excavated,evolved and analyzed by transcriptome.The expression of ACS genes were analyzed by qRT-PCR compared with the previously identified CS gene in order to investigate the relationship between the expression of ACS gene and capsaicin synthesis in peppers,and to infer the role of ACS genes in capsaicin synthesis pathway.The results are as follows:(1)The capsaicin content in the fruits of C.chinense,Hainan yellow lantern and Bhut jolokia were determined.The results showed that capsaicin accumulated mainly in placenta tissues of both.With the development of pepper fruits,capsaicin content in Hainan yellow lantern fruit increased gradually,and accelerated in the later stage;while capsaicin content in Bhut jolokia fruit increased in the early stage of development and maturity,and decreased to a certain extent in the later stage.The capsaicin content of fully mature pepper was significantly higher than that of Hainan yellow lantern,while the capsaicin content of two kinds of capsicum was significantly higher than that of reported C.annuum fruits.(2)42 ACS genes were extracted from the genome of C.chinense and analyzed for their evolution and expression.The results showed that all ACS genes in C.chinense co ntained a conserved domain.In phylogenetic trees,these genes are divided into two bran ches.Four genes highly expressed in C.chinense fruits were screened by significant diff erence analysis.The IDs in the reported C.chinense genome database were CC.CCv1.2.s CCffold 1155.1,CC.CCv1.2.sCCffol 462.15,CC.CCv1.2.sCCffold 859.1 and CC.CCv1.2.sCCffold 1075.12,which were named CcACS1,CcACS2,CcACS3 and CcACS4 respectivel y.Besides,among the 42 ACS genes,7 constitutive expression genes,6 significantly hig h expression genes in roots,1 high expression gene in flowers and 6 low or no expressi on genes in organs and fruits were identified.(3)Four ACS genes were screened by qRT-PCR and compared with previously identified CS genes.The results showed that the expression of four ACS genes increased with the development of Hainan yellow lantern fruit.The expression of CcACS1 in placenta was significantly higher than that of the other three genes,and the expression of CcACS1 in placenta was significantly higher than that in seed and pericarp.With the development of Bhut jolokia fruit,the expression of ACS gene and CS gene in placenta increased first and then decreased.The expression of CcACS3 in placenta was significantly higher than that of other three genes,and its expression in placenta was significantly higher than that in seed and pericarp.The expression level of CcACS2 and CACS4 genes in the seed of Hainan yellow lantern and Bhut jolokia were higher than those in placenta.Similar to C.annuum,CS gene expression in placenta of two C.chinense fruits was significantly higher than that in seed and paericarp.(4)The correlation analysis of four ACS genes and CS gene expression showed that the expression of CcACS2,CcACS3,CcACS4 in Hainan yellow lantern at different developmental stages were positively correlated with the expression of CS gene.Compared with the expression of CS gene,the expression of CcACS1 in Hainan yellow lantern pepper was slightly higher in the trans-chromogenic stage than in the developmental stage.so its expression was positively correlated with CS,but not significant.The expression of four ACS genes was positively correlated with CS in Bhut jolokia.To investigate the capsaicin content in different tissues of mature pepper fruits,the expression of CcACS1 and CcACS3 was positively correlated with the expression of CS gene in Hainan yellow lantern,while the expression of CcACS3 and CS gene in Bhut jolokia was positively correlated,but not significant.In this study,four ACS genes that may be involved in capsaicin synthesis in C.chinense were screened from 42 ACS genes by mining systematicly and expression analysis.The results showed that CcACS1 was most likely to participate in capsaicin synthesis in Hainan yellow lantern with relatively low pungency,while CcACS3 was the most likely to participate in capsaicin synthesis in Bhut jolokia with high pungency.Therefore,this study provides a molecular basis for further elucidating the mechanism of capsaicin synthesis,and provides a new gene target for molecular breeding with capsaicin content as the breeding objective.