Genetic Transformation Of Catharanthus Roseus With Dat And Str Genes And Promoter Analysis

The medicinal plant Madagascar Periwinkle (Catharanthus roseus)possesses a large number of terpenoid indole alkaloids (TIAs) in which over130compounds have been isolated and identified, such as vindoline, catha-ranthine, vinblastine, and vincristine. However, the contents of most TIAs inC. roseus are low and the most valuable dimeric alkaloids, vinblastine andvincristine, are difficult to be produced by cell culture and hairy root culturesystems. At present, the semi-synthetic process is normally employed to fab-ricate vinblastine and vincristine based on the extraction of relatively highabundance of vindoline and catharanthine in C. roseus. It is very difficult forcommercial production of important TIAs such as vinblastine by chemicalsynthesis due to the complexity of TIAs structures, low-output and high-costfor synthesis. Therefore, it is necessary to find ways to improve the contentof important TIAs in C. roseus and metabolic engineering strategy may beone of the choices. In this study, Agrobacterium tumefaciens-mediated transformationsystem of C. roseus was establishment, and the key genes involved in theTIAs biosynthetic pathway, dat and str, were transferred and overexpressedin C. roseus plants. Moreover, in order to study the expression regulation ofdat gene, the promoter of dat was isolated and analyzed, and the elementsinduced by MeJA were determined. The major results are as follows:(1) An efficient Agrobacterium-mediated transformation system of C.roseus was establishment. The influential factors of transformation were op-timized systematically and the optimal transformation conditions were ob-tained: using the hypocotyls as explants, sonication treatment for10minuteswith80W power, A. tumefacien infection for30minutes with OD6000.8andco-cultivation for2days in1/2MS medium containing100μmol/L acetosy-ringone. Lasting total,12independent transgenic C. roseus plants containingGUS gene were obtained, and the transformation frequency was among1.11%~11.11%.(2) Monovalent and bivalent expression vectors (pCAMBIA2300-dat,pCAMBIA2300-str-dat) containing the rate-limiting enzyme genes in theTIAs biosynthetic pathway in C. roseus, dat, and dat+str, were constructed.Forty independent transgenic C. roseus plants containing dat gene and10in-dependent transgenic C. roseus plants containing dat+str genes were obtained through Agrobacterium-mediated transformation. The transgenic plants werefurther analyzed by real-time PCR, and the results showed that the foreigngenes were overexpressed in transgenic C. roseus plants. The contents ofvindoline, catharanthine and vinblastine in transgenic C. roseus plants weredetermined by HPLC, and high TIAs-yielding plants were screened out fromtransgenic plants. Compared to the control (non-transgenic plants), the con-tents of the three TIAs including vindoline, catharanthine and vinblastine intransgenic C. roseus plants were significantly enhanced, with the highestoverexpressing dat gene containing1.92-fold of vindoline,1.42-fold of ca-tharanthine and16.70-fold of vinblastine, and the highest overexpressingdat+str genes containing2.48-fold of vindoline respectively,3.45-fold of ca-tharanthine and17.13-fold of vinblastine.(3) The promoter of dat gene (1773bp) was isolated using genomicDNA walking. The transcription start site of dat gene was determined by5’-RACE. The result showed that the transcription start site was A,13bpupstream of the ATG cordon. Sequence analysis showed that the distancebetween putative TATA-box and transcription start site was34bp. Severalcore fragments were identified, which are homologous to the cis-acting ele-ments of other plants and of great importance for promoter function. Thepromoter fragments with5’deletions and gain-of-function deletions were fused with GUS reporter gene, and their expressions were analyzed by tran-sient expression in C. roseus cell suspensions. With the fluorometric GUSassays in the suspension cells culture, it was found that the promoter regionfrom-252bp to-120bp was necessary for the regulation of the dat geneexpression. The TGACG motifs from-1112bp to-808bp in dat promoterwere identified, which may play an important role in methyl jasmonate re-sponse of dat gene.