Study On The Transformation Of AnANR Gene With Proanthocyanidins To S.Guianensis Cv Reyan No.2

Stylosanthes is an excellent tropical forage which is widely planted in the south of China and native to tropical regions of South America and Africa. Its traits including high yield, strong adaptability, nutritious value and contain a variety of vitamins and amino acids, the crude protein content is more than15%in the DM. High protein content of legume forage may cause digestive diseases of ruminants (such as cattle and sheep). Currently, in the breeding of Stylo, we have payed little attention to the application of improving condensed tannin content and protein utilization of forage legumes.In this study we have extracted and measured proanthocyanidins of12different species of Stylo with vanillin method. The results showed that S. guianensis cv. Reyan No.2with low content of proanthocyanidins, therefore we take S. guianensis cv. Reyan No.2as materials, and take the0.5cm young stem sections without bud as explants in the gene transformation, and find the regeneration system with optimized callus induction of Stylo. Meanwhile,we extracted RNA from’Alabama’and transcribed into cDNA, obtained proanthocyanidin biosynthesis gene AnANR full-length by PCR and plastic cutting technology, and construct expression vector CaM35S-GFP-AnANR, AnANR has been transformed into S. guianensis cv. Reyan No.2by Agrobacterium EHA105. We improved some factors of genetic transformation system ultimately and obtained a number of transgenic plants, Then PCR and RT-PCR has been taken to check transgenic plants. At last, we have extracted and measured proanthocyanidins of stylos with positive clones and CK controls, compared the content of proanthocyanidins after transformation.The main results are as follows:(1). The content of proanthocyanidins in12different species of Stylo were less than1%, none of them exceed the safe range of3%-5%, S. guianensis cv. Reyan No.2has less content than other10species, only slightly higher than one.(2). Improved regeneration system with young stem sections without bud as explants in the gene transformation. The best callus induction medium of S. guianensis cv. Reyan No.2is:MS+2,4-D2.0mg/L+KT4.0mg/L+Sucrose30g/L+7.5g/L Agar powder pH6.0, the composition for buds differentiation medium is:MS+6-BA0.2mg/L+NAA0.1mg/L+Sucrose30g/L+7.5g/L Agar powder, pH6.0, the root inducible medium is:MS+IAA0.5mg/L+Sucrose30g/L+7.5g/L Agar powder, pH6.0.(3). The optimum condition for genetic transformation of S. guianensis cv. Reyan No.2by Agrobacterium were:the usage amount of carbenicillin is500mg/L, the usage amount of hygromycin is15mg/L; the OD6oo of Agrobacterium concentration was0.8; (4). the submersion time of young stem with Agrobacterium was15min.(5). Using the technology of PCR and plastic cutting to get full-length of AnANR gene.Vector CaM35S-GFP was cut with Sac Ⅰ and BamH Ⅰ, inserted AnANR. gene into multiple cloning region which is under the control of CaM35S promoter to complete the plant expression vector construction which is named CaM35S-GFP-AnANR..(6). The positive transgenic stylos should be step-drilling for a week, and also need to take hydroponics of50d off before transplanting to nutrient soil. This two steps of transplanting increased the survival rate of transgenic stylos. Take five positive clones of transgenic stylos for sample randomly, then extracted and measured the contents of proanthocyanidins. The results showed that the content of proanthocyanidins in control group was0.225%, while the content of proanthocyanidins in positive transgenic stylos were0.263%-0.298%,which were slightly higher than that of control, we concluded that over-expression of AnANR gene from anthurium in S. guianensis cv. Reyan No.2may increase proanthocyanidins contents. This results supply reference for culturing new variety with high proanthocyanidins through gene engineering.