Cloning And Expression Of The Key Gene Of Capsaicin Biosynthesis AT3 And Its Regulatory Mechansim

Capsaicinoid(Capsaicinoid)is a unique secondary metabolite of Capsicum plants,which has broad application prospects and extremely high production value in food addition,medical care,biological control and other aspects.However,as a major producer of raw materials of capsicum,China has been unable to solve the problem of low production cost of capsaicinoid in the industrial production of capsaicin,rectify its reasons,different The content of capsaicin in cultivated capsicum is too different,C.Chinese type of capsicum with high spicy degree can not be widely cultivated as short-day plants in China,especially in the northern region,while C.anuum type of Capsicum widely cultivated in China has low capsaicin content and can not meet the production needs,while traditional genetic breeding can not cultivate high-yielding and high-spicy varieties,so mining different cultivated species of spicy The similarities and differences of key genes in capsaicin biosynthesis between peppers and the analysis of the mechanism of action regulating their expression are of great significance for improving the capsaicin content in high-yield peppers by gene technology.The AT3 gene encodes capsaicin synthase CS.Although it has been widely proved to be the most critical gene in the capsaicin biosynthesis pathway,the amount of its expression determines the amount of capsaicin in Capsicum fruit,whether there are differences in the AT3 gene and its promoter at the base level between different cultivated species of capsicum,whether this difference leads to differences in the degree of Capsicum spicy and AT3 gene.The expression is regulated by those factors,and such studies are not comprehensive.In this study,the AT3 gene and its promoter of different cultivars of pepper were cloned,compared and analyzed by bioinformatics.Through screening and expression analysis of genes related to capsaicin synthesis,the expression analysis of genes related to capsaicin synthesis in pepper fruit treated with exogenous hormones was carried out,and the results were asfollows:1.Clone to get C.chinese?C.frutescens and C.anuum Three types of Capsicum at3 gene,have different cDNA sequences,DNA sequences and promoter sequences.It is found that there are only discrete individual base differences in base level and amino acid level among the three genes.Bioinformatics analysis shows that the proteins encoded by the three genes are not different in functional properties,while the at3 promoter of C.chinese type capsicum is better than the other two types The at3 promoter in Capsicum has 10 bp more base segments in the sequence,and the number of at 3 promoter in CAAT box,TATA box and TCT motif is different from that in other two types of capsicum,which may be one of the reasons for different at 3expression in different cultivated capsicum.2.The expression of AT3,CAJMYB,CAFMYB,CADMYB and CEFMYB in different pepper stem and leaf fruits was obtained by qPCR experiment.According to the specific expression characteristics of AT3 gene in pepper fruits,three MYB transcription factors,CAJMYB,CAFMYB and CADMYB,which can have higher expression levels in three types of pepper fruits,were screened out,and there was a possibility of regulating AT3 gene expression in all three types of pepper fruits.3.The capsaicin content in C.Chinese type was the highest,followed by C.frutescens type,and the lowest in C.anuum type.The capsaicin content in the three types of Capsicum fruits increased gradually with the growth and development of the capsicum fruits until the end of the overripening period.The results of qPCR using the same material showed that AT3 gene expression in C.Chinese type pepper was the highest,which was in expression of CAJMYB gene was the highest in C.Chinese type pepper,but there was no significant difference between the other two types of pepper,in which the expression in C.Chinese type pepper was about 1.5 times that in C.anuum type pepper and 1.5 times that in the other two types of pepper.The expression of CADMYB gene in C.Chinese type pepper was the highest,about 1.6 times of that in C.frutescens type pepper,2.5 times of that in C.anuum type pepper,and increased with fruit development in single pepper.Therefore,CAJMYB and CADMYB are important transcription factors regulating AT3 expression.4.Three types of Capsicum plants in juvenile stage were sprayed with hormones SA,MeJA,SA+MeJA,BR and H3BO3,respectively,and sprayed with clear water as control.When the fruits were sampled once during the coloration and maturation stages,qPCR experiments were carried out.It was found that the exogenous hormones SA,MeJA and BR could increase the expression of capsaicin biosynthesis-related genes AT3,CAJMYB and CADMYB in Capsicum fruits,but SA The expression of capsaicin biosynthesis-related genes in pepper fruits did not change when + MeJA was mixed.H3BO3 treatment could increase the expression of AT3 gene in pepper fruits,but had no effect on the expression of CAJMYB and CADMYB.In addition,when capsaicin biosynthesis-related genes were regulated by exogenous hormones,their expression levels in the fruit color-changing and ripening stages were higher than those in the control group,and the increase in the expression of genes related to the color-changing stage was often greater than that in the ripening stage.In addition,the expression of capsaicin biosynthesis-related genes AT3,CAJMYB and CADMYB increased by about 30%-60% in SA-treated fruits among different peppers,while the expression of related genes AT3,CAJMYB and CADMYB in C.anuum type pepper fruits after MeJA and BR treatment changed greatly,19-21 times as compared with the control group,while C.chinese and C.Frutescens expression of related genes in frutescens type pepper fruit was only 1.5-2.5 times of that in the control group,and the expression of AT3 gene in C.anuum type pepper fruit after H3BO3 treatment was about 5.1 times,C.chinese,C.Frutescens expression of AT3 gene in frutescens type pepper fruit was only 1.2-1.6 times of that in the control group,indicating that the regulation of capsaicin biosynthesis-related genes by SA was not affected by pepper cultivars,while the regulation of MeJA,BR,H3BO3 on specific pepper cultivars C.annum type was more obvious,which could greatly increase the expression of related genes.