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Molecular Cloning Of Cinnamic Acid4-hydroxylase Gene,4-coumarate: Coa Ligase Gene And Chalcone Synthase Gene In Mulberry(morus Multicaulis) And Expression Analysis Of Them Among Different Germplasm Resources

Posted on:2015-08-11Degree:MasterType:Thesis
Country:ChinaCandidate:M F HuangFull Text:PDF
GTID:2180330422988613Subject:Biochemistry and Molecular Biology
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Mulberry (Morus L.) is a kind of important ecological and economic tree. It is used asthe forage of silkworms, but also as important materials of food and medicine. Flavonoid isone of the main active ingredients of mulberry. We have isolated and identified severalflavonoid compounds from mulberry leaves, and found that the flavonoid content ofmulberry leaves is very high. In this research, we cloned three key enzyme genes C4H,4CLand CHS regulating flavonoid biosynthetic pathway in mulberry leaves, analysed theexpression of the above genes among different mulberry germplasm resources usingreal-time quantitative PCR, compared the expression level with the total flavonoids contentof mulberry leaves. The results can provide some references for controlling the food andmedicinal materials quality and breeding high flavonoid content mulberry varieties bymolecular breeding methods.The main results were summarized briefly as follows:1、Cloning and Analysis of Cinnamic Acid4-hydroxylase Gene (MmC4H) in Mulberry(Morus multicaulis)Cinnamic acid-4-hydroxylase (C4H) is one of the key enzyme in pheny1propanoidmetabolic pathway which affects the biosynthesis of flavonoids and other secondarymetabolites. The cDNA encoding C4H was cloned through the methods of RT-PCR andRACE from mulberry (Morus multicaulis) younger leaves, which was designated asMmC4H (GenBank NO. KJ013408). It is1794bp long and has a complete open readingframe (ORF) of1518bp, which encodes a protein of505amino acid and the proteinsequence contains a plant P450conserved domain. Comparing the amino acid sequencewith the6other corresponding genes from NCBI nucleic acid and protein database, theresult shows that it shared high similarity (above86%).2、Cloning and Analysis of4-Coumarate:Coenzyme A Ligase Gene (Mm4CL) in Mulberry(Morus multicaulis)4-coumarate: CoA ligase is a key enzyme in the phenylpropanoid metabolic pathwayfor monolignol and flavonoid biosynthesis. The cDNA encoding4CL was cloned throughthe methods of RT-PCR and RACE from mulberry (Morus multicaulis) younger leaves,which was designated as Mm4CL (GenBank NO. KJ013409). It is1890bp long andcontains a105bp5’-UTR (untranslated region) and a132bp3’-UTR. It’s opening frame(ORF) is of1653bp, which encodes a protein of550amino acid with apredicted molecularweight of60.30KD and an isoelectric point of5.64. Comparing the amino acid sequencewith the6other corresponding genes from NCBI nucleic acid and protein database, theresult shows that it shared high similarity (above76%). 3、Cloning and Analysis of Chalcone synthase Gene (MmCHS) in Mulberry (Morusmulticaulis)Chalcone synthase (CHS) is a key enzyme in flavonoids synthesis pathway,which catalyzesthe first rate-limiting step of flavonoid biosynthesis. The cDNA encoding CHS was clonedthrough the methods of RT-PCR and RACE from mulberry (Morus multicaulis) youngerleaves, which was designated as MmCHS (GenBank NO. KJ013407). It is1505bp long andcontains a116bp5’-UTR (untranslated region) and a207bp3’-UTR. It’s opening frame(ORF) is of1182bp, which encodes a protein of393amino acid with apredicted molecularweight of42.91KD and an isoelectric point of6.15. Comparing the amino acid sequencewith the6other corresponding genes from NCBI nucleic acid and protein database, theresult shows that it shared high similarity (above87%).4、Analysis of MmC4H’s、Mm4CL’s and MmCHS’s Expression among Different MulberryVarietiesThe Real-time quantitative PCR analysis and mulberry leaf flavonoids contentdetermination showed that expression level of MmC4H、Mm4CL and MmCHS weresignificant different among mulberry varieties, and their expression level also have certaincorrelation with mulberry leaf flavonoids content. The research may provide somereferences for the breeding of new mulberry varieties with high content of flavonoids.
Keywords/Search Tags:Morus L., Cinnamic acid-4-hydroxylase, 4-coumarate, CoA ligase, Chalconesynthase, Gene cloning, Gene expression, Flavonoid content
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