Over-expression Of Atpap1Gene In Tobacco And Regulation Of Anthocyanin Biosynthesis

To achieve the accumulation of anthocyanin in tobacco and tobacco glandularhairs, and explore applied and research potential of anthocyanins on tobacco qualityimprovement.this article cloned arabidopsis MYB family of transcription factors PAP1,and construct overexpression vector of constitutive plant, using the method of Tiplasmid-mediated to transform tobacco, achieving PAP1gene heterologousoverexpression in tobacco and tobacco glandular hairs, getting "Red Tobacco" whichaccumulate anthocyanins. Meanwhile, the CYP71D16promoter sequence ofglandular hairs specifically expressed genes in tobacco was cloned. The system ofglandular hairs specific expression was established. the specific gene expression ofPAP1in glandular hairs was achieved.and the "red glandular hairs tobacco" wasobtained. The foundation for the development and research of biological reactor oftobacco glandular hairs was established. The main findings are as follows：PAP1gene is constitutively expressed in tobacco：Cloning PAP1gene wasinserted into pCAMBIA1391vector, and the plant constitutive expression vectorpC1391-35S-PAP1was constructed. The leaf discs of tobacco (Nicotinan tabaciumT.I1068) were transformed with the gene via Agrobacterium-mediated transformation,red buds were regenerated on rooting medium MS+NAA0.1with2mg/L PPT, l.5mg/L6-BA,0.1mg/L IAA and then becomed regenerated plants. Molecular testing provedPAP1gene had achieved the integration and expression in tobacco. The accumulationof anthocyanin in the leaves of transgenic plants is induced by the glare.The system of specific expression in glandular hairs of tobacco was established:The promoter sequence of the gene CYP71D16that is expressed specifically intobacco glandular trichomes is cloned, and inserted it into the plant expression vectorpCAMBIA1391, then the tobacco glandular trichomes specific expression vectorpCAMBIA1391-CYP-GUS was built. Transformed the plasmid into tobacco viaAgrobacterium-mediated transformation and obtained resistant plants by screening onthe MS differentiation medium with6mg/L hygromycin (Hyg), l.5mg/L6-BA,0.1mg/L IAA. The molecular testing proved that the GUS gene was integrated andtranscrbed in the genome of tobacco. Histochemical test showed that the glandular trichomes were blue on the surface of transgenic plants, which is the specific reaction,indicating that the GUS gene were expressed specifically in tobacco glandulartrichomes successfully.Specifical expression of PAP1gene in the tobacco glandular trichomes:construction of pCAMBIA1391-CYP-PAP1expression vector, using the leaf disk totransform tobacco T.I1086. This paper got resistant plants on the differentiation MSmediumwith9mg/L hygromycin (Hyg), l.5mg/L6-BA,0.1mg/L IAA. The moleculardetection indicated that PAP1gene was consolidated and transcripted in the tobaccogenome, and tobacco glandular trichomes were found appeared lighe red by themicroscope, initially indicates that anthocyanin is specifically accumulated in thetransgenic tobacco glandular trichomes.According to a comparative analysis of transgenic plants, aiming atconstitutively expressing“red tobacoo” and glandular hairs specific expression“redglandular hairs tobacco” by PAP1,which analyzed morphological and chemicalcomposition. LC/MS analysis showed that “red tobacoo” leaves contained high redcyanidin (Cya) and low pelargonidin(Pel) and “red glandular hairs tobacco” leavesonly contained small amounts of cyanidin (Cya), however, CK T.I1068didn’t containanthocyanins in leaves. Compared “red tobacoo” with transgenic control byobserving morphology, its phenotype changed greatly,leaves were light red, stemcurved and leaf margin curved. However, red glandular hairs tobacco plants didn’’tshow obvious morphological difference, except that part of the glandular hairs werewere light red.