Isolation And Expression Analysis Of Gene Encoding The Key Enzymes In Phenylpropanoid Metabolic Pathway In The Flue-cured Tobacco

Polyphenols were closely associated with the level of tobacco, fragrance and taste and tobacco safety. Polyphenol mainly derived from phenolic metabolism. Studies have shown that phenylalanine ammonia-lyase, cinnamic acid-4-hydroxylase and p-coumaric acid-4-ligase are the key enzymes in the metabolism. They made to synthesis polyphenols, which affects the quality of tobacco. But it have not been reported about their molecular biology characteristics and time-space expression characteristics.To further study the molecular mechanism of polyphenols biosynthesis, we did this research. The cDNA encoding full-length of Ntpall、Ntpal2, Ntpa13、Ntpal4、Ntc4hl、Ntc4h2、Nt4cl1and Nt4c12genes were cloned from two kinds of flue-cured tobacco varieties Honghuadajinyuan and K326, which had significant genetic differences.Its nucleotide sequences and encoded amino acid sequences and the protein functional domains were analyzed by bioinformatics methods. In this paper,we studied tissue expression analysis of Ntc4hl、Ntc4h2、Nt4cll and Nt4cl2genes in mature tobacco varieties Honghuadajinyuan and K326, using technology of fluorescence quantitative real-time PCR(QRT-PCR). We also carried out the research of expression properities of Ntc4hl、Ntc4h2、Nt4cl1and Nt4cl2from tobacco leaves in different growing stages. In addition, the enzyme activity of C4H and4CL from different position leaves were studied.Our goal is to study the gene function from the perspective of gene regulation and to improve the synthesis of polyphenols.Thereby it can enhance the quality of tobacco in production. The main results are as follows:(1) In this study, four ordinary tobacco genes Ntpal1、Ntpal2、Ntpal3and Ntpal4were cloned and isolated.They four have the same length, but their open reading frame were respectively2148bp,2139bp,2139bp,2154bp, and they could encode715,712,712and717amino acids respectively. Ntpal3in two flue-cured tobacco varieties are identical.(2) In this study, two ordinary tobacco genes Ntc4hl and Ntc4h2were isolated and cloned. They two also have the same length and the same open reading frame1518bp, and they could encode505amino acids. The similarity of Ntc4hl between the two species was99.87%, while the similarity of Ntc4h2between the two species was99.93%. The two homologous genetic differences among varieties is small. Ntc4hl and Ntc4h2in Two varieties have a98.63%difference.(3) Nt4cll and Nt4cl2genes were also isolated and cloned from two tobacco varieties. Their open reading frame were respectively1644bp and1629bp, and they could encode547and502amino acids respectively. As for homologous gene Nt4cll, its similarity between the two cultivars was99.94%, while the similarity of the two genes Nt4cl2between cultivars is100%.(4) Ntc4hl, Ntc4h2、Nt4cll and Nt4cl2were all expressed in different tissues. The two homologous genes Ntc4h1and Ntc4h2in different tissues from Honghuadajinyuan have similar expression characteristics, the expression of the xylem and phloem was significantly higher than in other tissues.The two homologous genes Nt4cll and Nt4cl2in different tissues from Honghuadajinyuan has the same expression properities as Ntc4hl and Ntc4h2.Also, it basically showed higher expression trend in xylem than in phloem. However, as for each of the two homologous genes Ntc4h and Nt4cl, the expression levels in different tissues from K326were inconsistent with that in Hongda.In addition, Ntc4h2and Nt4c12in K326showed higher expression of xylem and phloem than in any other tissues.(5) The expression characteristics of two homologous genes of Ntc4h were similar in the middle leaves of two different varieties, and the leaves were taken from tobaccos in different growing stages. The level of expression in topping stage and technical maturity stage was significantly higher than that in fast growing period.However, two homologous genes of Nt4cl in Hongda showed another situition, that is, the level of expression in topping stage and technical maturity stage was significantly higher than that in fast growing period, but it did not show like this in K326.(6) C4H enzyme activity showed different in different level leaves, which were from Hongda and K326. In technical maturity stage, enzyme activity of C4H from Hongda were as follows:middle leaves> upper leaves> lower leaves. While that in K326is:upper leaves> lower leaves> middle leaves. Enzyme activity of4CL from Hongda and K326were both showed that middle leaves> lower leaves> upper leaves, which was consistent with expression of Nt4cll in different tissues.