Study On The Key Genes And Their Regulation Functions In The Terpenoid Indole Alkaloids Biosynthetic Pathway In Catharanthus Roseus L. G. Don

The biosynthetic pathway of terpenoid indole alkaloids (TIAs) in Catharanthusroseus cells is a complex metabolic network which is composed of a lot ofbiochemical reactions and controlled strictly. The number of cloned structural geneand transcriptional gene has been over30till now, and the research work on theenzymes and the genes involved the TIAs biosynthesis have been carried out fordecades. The rough sketch of TIAs biosynthetic pathway in C. roseus is clear,however, it is still unclear that which enzymes in the TIAs Biosynthetic Pathway arethe rate-limiting enzymes. Until now, there is no any report on the comparison of geneexpression involved in TIAs biosynthetic pathway. According to the situation, wedesigned and conducted the experiments on the comparison of gene expressioninvolved in TIAs biosynthetic pathway. Young leaves from6-week-old sterile C.roseus plants were harvested for hairy root induction. Six enzymes (DXR, G10H,STR, TDC, DAT, PRX1）in the TIAs biosynthetic pathway and two transcriptionalfactors (ORCA2and ORCA3) were choien to be targets in our research. Eightmonovalent plant expression vectors (pCAMBIA1304++gene) and one biovalent plantexpression vectors (pCAMBIA1304++Orca2+Orca3) were constructed whichcontained the genes: dxr, g10h, tdc, str, dat, prx1, orca2, orca3, respectively,according to the sequences from NCBI GenBank. The vectors were separatelyintroduced into Agrobacterium tumefaciens strain C58C1which carries the Riplasmid from A. rhizogenes strain A4and then were used to transform leaf explants ofC. roseus. The transformed roots were inducted and the independent root clones weresubcultured on solid MS cefataxine (250mg/l) agar medium firstly and then in liquid1/2B5medium. After selecting the transformed hairy roots growing on the selectivemedium, the DNA and RNA were extracted from the hairy roots for moleculardetection and fluroscent real-time quantitative PCR (FQ-PCR), respectively. Finally,the roots were confirmed the presence of rol B and rol C genes, the hygromycin (hpt)resistance gene, and the target gene in the transformed roots by PCR analysis. FQ-PCR were used for detecting the relative expression of the gene transformed inthe hairy roots in comparison to control samples. High-performance liquidchromatography (HPLC) was used for detecting catharanthine and vindoline contentin the hairy roots and the results were analyzed.Our results were analyzed and the conclusions were listed as follows:1. DXR、STR are the key enzymes of up-stream and middle-stream in the TIAsbiosynthetic pathway, respectively. After detecting the catharanthine and vindolinecontent in the all hairy roots by HPLC, the results show that all samples increase theaccumulation of TIAs in comparison to control lines. Among them, the root linestransformed with str single-gene accumulate the highest catharanthine content（6.579±0.711mg/g DW). The root lines transformed with tdc or dat single-gene accumulatethe lowest catharanthine content (3.177±0.623mg/g DW、3.488±0.696mg/g DW,respectively), which are as lower as the control line (2.77±0.652mg/g DW). Theaverage vindoline content in the roots with overexpressing dxr or prx1gene are higherthan the root samples overexpressing g10h, str, tdc, dat gene, respectively. Thevindoline content in the roots transformed with dat or tdc gene are lower in allsamples (0.041±0.0073mg/g DW、0.044±0.0091mg/g DW), which is almostequealed to the control samples (0.0375±0.007mg/g DW). Therefore, we concludethat DXR and STR are the key enzymes of up-stream and middle-stream in the TIAsbiosynthetic pathway. G10H is not important as expected and it may functions undersome conditions.2. PRX1is a rate-limiting enzyme down-stream in the TIAs biosynthetic pathway.The transformed hairy roots of orca3single-gene were detected by FQ-PCR, therelative gene expression of prx1gene is the highest among all structural genesdetected, average relative gene expression is12.18±1.442. The high expressionsuggests that regulator ORCA3can control the biosynthesis rate of down-stream inthe TIAs biosynthetic pathway by regulating the expression of prx1gene. On theother hand, catharanthine and vindoline content in the root samples transformed withprx1single-gene detected by HPLC are higher than the controls significantly, andespecially the vindoline content is the highest among all the samples (0.1043±0.017 mg/g DW). The results above demonstrate that PRX1is a rate-limiting enzyme ofdown-stream in the TIAs biosynthetic pathway.3. G10h gene is the target gene regulated by ORCA2transcriptional factor. Eightroot samples of overexpressing orca2single-gene were analyses by FQ-PCR and therelative expressions are almost ideal, the average amount is1.642with standard erroronly S=0.288. In addition, eight root samples of overexpressing orca3single-genewere analyses, the average expressions amount is0.57±0.093. The results abovedemonstrate that g10h is regulated by ORCA2, but not by ORCA3.4. The expression of lamt gene and sls gene are controlled by both regulatorORCA2and ORCA3. The transformed hairy roots of orca2and orca3gene wereanalyzed by FQ-PCR, the relative expression of lamt gene is2.8±0.634and7.437±0.632, while the relative expression of sls gene is3.55±0.76and3.265±1.06,respectively. The data above illustrates the expression of lamt gene and sls gene iscontrolled by the two regulators and suggest that LAMT and SLS may be important inthe TIAs biosynthesis.5. The expression of hmgr gene is regulated by ORCA3. The results analyzed withFQ-PCR showed that relative expression of hmgr in transformed hairy root lines oforca2and orca3is1.37±0.42and6.64±1.21. The facts demonstrate the expressionof hmge is strongly controlled by ORCA3. It suggests us that ORCA3not onlycontrols the MEP pathway up-stream in the TIAs biosynthetic pathway so that tosupport the TIAs biosynthesis, but also controls the MVA pathway effectively andsimply by controlling the expression of hmgr gene which functions to control theMEP pathway and the MVA pathway coordinately.6. The hairy roots analysis by HPLC overexpressing orca2single-gene, orca3single-gene and orca2/orca3double-gene approved that the catharanthine andvindoline content are higher than that of the control lines, respectively.Transformation with double-gene can get more the TIAs content compared to thesingle-gene transformation.