Studies On Gene Expression Of Anthocyanin Biosynthetic Pathway And AMYB Expression In Narcissus Tazetta L. Var. Chinensis Roem

Chinese narcissus (Narcissus tazetta var. Chinensis) belongs to perennial herb ofAmaryllidaceae and one of famous traditional flowers in China. The basicmechanism in floral color formation has not yet been clearly explained on Chinesenarcissus. In this study, αMYB gene was cloned and transient expressed in flowers ofZhangzhou narcissus by particle bombard. Zhangzhou narcissus 'Jinzhanyintai' wasused as test materials, daffodils "Pink charm" as the control, the cDNA conservedfragments of the anthocyanin metabolic pathway-related genes were cloned andtheir expressions in different developing times and parts of narcissus flowers byRT-PCR detection were performed. And aMYB was introduced into Zhangzhounarcissus using the optimal transformation system. The results were as follows:1. Using the method of homologous cloning, the conserved regions of DFR, ANSand FLS were obtained.The cDNA fragment of DFR is449bp and encoding149amino acids (GenBank accession No. JN712435). The cDNA fragment of ANS is291bp and encoding96amino acids (GenBank accession No. JN688963). The cDNAfragment of FLS is249bp and encoding83amino acids (GenBank accession No.JQ013783). Expression of CHS, CHI, F3H, DFR, ANS and FLS in differentdevelopmental stages and different parts of chinese narcissus was studied bysemi-quantitative RT-PCR, the results showed that CHS, F3H, ANS and FLS wereobserved expression both in petal and paracorolla, but the expression of CHI and DFRwere little or weak. We suggest that CHI, DFR and FLS are the key genes of inanthocyanin metabolic pathway of Chinese narcissus, little expression of CHI andDFR in the early developmental stages of flowers in chinese narcissus may be the keyreason why Chinese narcissus doesn't have anthocyanin compounds in the flower.2. The improved Trizol method can be very good for RNA extraction of α plant,high quality RNA was obtained and beneficial to subsequent steps.3. The GUS gene in pC1301was replaced by αMYB gene, plant expression vectorpC1301-αMYB was successfully constructed. Red spots were observed in both petalsand paracorollas of chinese narcissus by transient expression of αMYB gene, whichproved that exogenous regulatory factor αMYB had an impact in regulation ofanthocyanin metabolic pathway and structure genes, anthocyanins was accumulatedand color change was produced.4. αMYB gene was transformed into chinese narcissus by Agrobacterium- mediated transformation using the established regeneration system. Resistant smallbulbs were obtained.