Environmental Regulation And Gene Expression Analysis For Biosynthesis Of Terpenoid Indole Alkaloids In Catharanthus Roseus

Alkaloids are nitrogen-containing basic compounds in plants and constitute one of the largest groups of natural secondary metabolites. People have been of great interest in alkaloids because of their pharmacological activities. Catharanthus roseus (C. roseus) contains important anti-tumor terpenoid indole alkaloids (TIAs), such as vinblastine and vincristine. It was made certain the mode plant in study the pathway of secondary metabolites, for the cause of it has astracted many peoples attention in biopharmacy at present. Most of these TIAs are produced at low levels in the natural plants and are difficult to be chemically synthesized due to their complex structures. There remains a need to increase alkaloids production rates for commercial exploitation.Catharanthus roseus (C. roseus) is belonged to liking light plant, and grown favoritely under high temperature and high humidity environment or tolerance of semi-shade condition. In recent years, researchers who studied the effects of environmental factor on C. roseus found that TIAs accumulation were related to environmental factor. But, environmental regulation and gene expression analysis for biosynthesis of terpenoid indole alkaloids in C. roseus are reported less. This paper studied the effects of red films on the contents of pigment and alkaloids, and on the expression of biosynthesis pathway genes in the leaves of C. roseus aseptic seedlings. The further correlation analysis between alkaloids content and the genes transcriptional level were studied. In addition, the experiment of the contents of pigment and alkaloids, and on the expression of biosynthesis pathway genes in the leaves of C. roseus seedlings under the same red films were taken, which illustrated that light intensity and light quality affected the alkaloids accumulation. On the other hand, with six kinds of PEG6000 concentration to imitate drought stress, we researched the changes of proline content and relative water content with the theory of plant physiological ecology. Then we selected the most suitable PEG6000 concentration and treated the leaves of C.roseus seedlings, The Purpose of our experiment was that discussing the relation of POD activities, genes expression and alkaloids content under PEG stress. The result is followed:(1) The contents of vindoline (VIN), catharanthine (CAT) and vinblastine (VBL) in the leave of C.roseus aseptic seedlings were measured by HPLC. The content of VIN and CAT firstly increase a little and then decrease compared to control. The content of VBL generated by its precursors VIN and CAT in leaves of C. roseus aseptic seedlings increase gradually and reached the highest on the 15th day in shade culture. SQ-RT-PCR method was employed to study the expression levels of 5 important genes(G10h, Tdc, Str, D4h and Dat), the results showed:G10h gene expression was not significantly changed, there were positive correlativity of the expression levels of these four genes (Tdc, Str, D4h and Dat) with accumulation of VIN and CAT under low light environment. So, we might speculate the accumulation of alkaloids in the leaves of seedlings.(2) The same red films shade C. roseus seedlings. The results showed:red the films induced a low light condition, but the low light environment was different from (1). There was correclations between alkaloids content and the genes transcriptional level (Tdc, Str, D4h and Dat) at certain time. The above results demonstrate that the shade treatment with red films accelerated the accumulation of VIN and CAT in the leaves of seedlings. In contrast to (1):the change of chlorophyll content was different from (1), and the change of carotenoids content was different from (1), which might regulate plant auxin in C.roseus and change the VBL content.(3) The research determined the content variety of proline in leaves of C.roseus seedlings with deal time under different PEG6000 concentration stresses, as well as the relative water content (RWC) were measured by the method of saturated water. The resules show that the developing trend of RWC is declining and rising, and the developing trend of Pro content is increasing slowly and increasing quickly with the development of stress time under the same PEG6000 concentration. By combination and optimization of various conditions, a sample preparation method from leaves of C. roseus was established.(4) POD activities were analyzed in the leaves of C. roseus under 35% PEG6000 concentration stress. The activities are increasing gradually compared to control, while is beneficial to the VBL biosynthesis from VIN and CAT. The content of VIN and CAT are decreasing, VBL content is increasing under drought stress. The results show that POD activities could regulate the transform from monomeric indole alkaloids to bis-indole alkaloids under osmosis stress. At the same time, the expression of Tdc, Str and Dat are higher than control in certain time under stress, the related enzyme are activated and indicate that alkaloids accumulation is tightly correlated with these genes expression in C. roseus.(5) Cell wall is a fundamental structure in plant, it is important to grow, stress and convey information in plant cell. Pectin methyl esterases (PMEs; EC 3.1.1.11) are extracellular enzymes that catalyse the de-esterification of methylesterified pectin. In this study we addressed the question of the physiological function of PME in plant development by cloning Pme gene that is expressed in leaves of C. roseus. PME as biological inductor regulate alkaloids biosynthesis, which might provide a new way for environmental regulation.