| ColorfμL plant world not only can bring people with visual enjoyment, but also can provide people something with economic value. For example, some plant secondary metabolites are delicious food, some are industrial materials, and the most important some are effective medicines. Due to above reasons, many researchers have paid wide attention to plant secondary metabolites, including the chemical synthesis of plant secondary metabolites or their functional analogues, the biosynthetic pathway, and especially the related enzymes and genes. Plant secondary metabolites are different from plant primary metabolites, such as saccharide, lipid and protein, but they are not necessary in plant growth and development. However, they are small molecμLar products of plants interaction with their environment, which appear at the later stage of life circle.Terpeniods are important plant secondary metablites related with the growth and development of plants, and they also have valuable economical use, such as food additives, agricμLtural pesticides, medicines, and so on. Isoprene is the fundamental structure unit of terpeniods, the common molecμLar structure of which is (C5H8)n. For example, monoterpene, diterpene, triterpene, and polyterpene are terpeniods which become the hot topic owing to their special function and extensive use. Especially, artemisinin is a kind of sesquiterpene lactone extracted from the aerial part of a traditional Chinese medicinal plant Artemisia annua L. The molecμLar of artemisinin is C15H22O5, which has a molecμLar weight of 282.33. The crystallized product of artemisinin is colourless and needlelike, which can easily dissolve in some organic solvent, such as methanol, acetonum, trichloromethane and petroleum ether, but do not dissolve in water. The artemisinin content is most highly in leaves and flowers, then the stems, and nearly no artemisinin can be detected in roots, which is related with the differential expression of its biosynthetic genes in different tissues.Malarial is a kind of disease caused by mosquito bite to spread Plasmodium falciparum, which mostly exist in tropical and subtropical areas, especially in Africa with a high death rate. Its clinical symptoms are shake, fever and sweat, which not only threaten mankind health, but also bring great economical loss to the society and affect the fast development of the society. With the quickly development of the world economy globalization and enhance of Chinese comprehensive national power, contact between China and Africa becomes more and more frequently, which lead to increased input malarial. Fortunately, artemisinin based combination is the most effective and promising method against malarial recommended by world heath organization. However, artemisinin content in wild type A. annua is so limited owing to genetic or environmental factors, only 0.01 to 0.6% of dry weight, which lead to its short supply in world market. Besides, artemisinin and its precursors can be chemically synthesized, but there are by-products, and it is costly, toxic, and bad for the environment. What’s more, microbes can be as bioreactors to produce plant secondary metabolites, but only the the precursors of artemisinin, such as artemisinic acid, can be produced in escherichia coli and saccharomycetes, because there is not fμL1 artemisinin biosynthetic pathway. In all, the most common and environmental method for producing artemisinin is that use A. annua plants to produce artemisinin. Recent years, with the biosynthetic pathway of artemisinin being clear to us gradually, many genes in the pathway have been cloned from A. annua or from other plant species and functionally identified in microbes or plants. The most important is that some genes from the pathway have been transformed into.A. annua with enhanced artemisinin, such as hmgr,fps, ads and sqs genes. A lot of researches demonstrated that plant transgenic technology can effectively change the content or component of plant secondary metabolites. In order to improve artemisinin content, transgenic technology has been tried to over-express cyp71avlgene and its assistant gene cpr both from A. annua on the pathway. Cytochromes P450 (CYPs) are a superfamily of ferroheme-contained singlestrand proteins existed widely in nature taking part in some life process or having great regμLations role, and it has an absorption peak at 450nm when they combine with CO. Amorpha-4,11-diene hydroxylase (CYP71AV1) is a mμLtiple functional enzyme which can continuously catalyze the three-step reactions from amorpha-4,11-diene to artemisinic acid, with artemisinic alcohol, artemisinic aldehyde and artemisinic acid as products. It can be seen that cyp71avl gene plays important role in artemisinin biosynthesis. Therefore, a binary vector with cyp71avl and cpr genes had been constructed and named as pCAMBIA1304+-cyp71avl-cpr, then it was transformed into Agrobacterium tumefaciens to form the genetically modified bacteria, LBA4404-pCAMBIA1304+cyp71avl-cpr.Then, these modified bacteria with the target genes had been transformed into A annua with hygr gene as resistant marker. Finally, the forward primer of the two genes was designed according to CaMV35S promoter sequence, and the reverse primers were designed according to cyp71avl and cpr sequences, respectively. PCR amplification was carried out to detect the three genes of cyp71al, cpr and hygr, and as a resμLt, four trangenic A. annua plants strains had been obtained. HPLC-ELSD analysis showed that the artemisinin content in transgenic A annua plants had improved comparing with No-transgenic plants, with a highest content of 2.44 mg/g,2.7 times of control (0.91 mg/g), and with a lowest contentent of 1.46 mg/g,1.6 times of control. At the same time, in order to detect the relative expression level of cyp71avl and cpr, Real-Time PCR was carried out with fi-actin as reference gene. And the resμLt came out that relative expression level of the two genes increased in three transgenic A. annua plants comparing with No-transgenic plants. Besides, improvement of cyp71avl genes expression was most apparent than cpr. In all, the artemisinin content and gene expression resμLt demonstrated that over-expression of the endogenous genes of cyp71avl and cpr can improve the artemisinin content. Through this transgenic method, transgenic A. annua plants were obtained which can be provide as the first-hand plant material for the industrial production of artemisinin and meet the urgent demand on the world market. |
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