Cloning And Functional Verification Of Vitamin E Synthesis-related Gene(MsHppd And MsHpt)and Vitamin E Content Determined Of Alfalfa(Medicago Sativa)

Alfalfa(Medicago Sativa L.)is a kind of high-quality perennial leguminous forage from the near east and central Asia which named as the king of grazing grasses.It contains the rich protein,vitamins,minerals and complete kinds of amino acids,also the good palatability,wide adaptability and stability productivity.Vitamin E in improving alfalfa nutritional quality and enhance the capacity of alfalfa salt tolerance has a very important role.Vitamin E is a natural antioxidant only synthesis by green plants,It's play an significant role of improve the nutrient and salt tolerance of Medicago Sativa L.The vitamin E(VE)content and main component of 16 alfalfa(Medicago Sativa L.)varieties were determined using high performance liquid chromatography(HPLC)technique in this study,analysis the correlations among total VE content in the distribution and the relationship between the VE content and the seed vigor.Cloning and analysis MsHppd and MsHpt from Medicago Sativa L.Construct and transfer the overexpression vector into Arabidopsis thaliana(ecotype columbia).Verification and conclud the function of this two genes by the content and distribution of vitamin E and the physiological indexes in transgeneticlines.The main results showed that:1.In this study,the vitamin E(VE)content and main component of 16 alfalfa.(Medicago Sativa L.)varieties were determined using high performance liquid chromatography(HPLC)technique.The analysis results showed that the total VE content of 16 varieties ranged from1.833 to 6.346 mg·(100 g)-1(FW),and the mean value is 3.281 mg·(100 g)-1(FW)(P<0.01).Zhong mu NO.1 contains the highest VE content of 6.346 mg·(100 g)-1(FW),whereas Aohan has the least content of 1.833 mg·(100 g)-1(FW).?-VE is the main component of total VE,?-VE take the second place,?-VE is the least part of total content.Analysis the result with the alfalfa plant areas we found that the alfalfa come from Huang-Huai-Hai Region of China have the hightest content of VE,range of variation 3.027?6.346 mg·(100 g)-1(FW),mean value 4.687 mg·(100 g)-1(FW)and the alfalfa come from Inner Mongolia Plateau have the least content of VE,range of variation 1.833?2.150 mg·(100 g)-1(FW),mean value 1.959 mg·(100 g)-1(FW).Correlation analysis indicated that the vitamin E have great influenc on the seed vigor and the root growth when germination.2.We got two gene MsHppd and MsHpt which have the important role in vitamin E synthetic by RACE,Bioinformatics analysis showed that MsHppd and MsHpt respectively contained 1308 and 1236 bases of the complete open reading frame,construct the overexpression vector by pBI121 vector,then transfer the overexpression vector into Arabidopsis thaliana(ecotype columbia).Verified and selected the positive line by Kan-resistance screening,PCR identification,GUS histochemical assays,southern blot and western blot.Results show that both of two genes successfully imported arabidopsis thaliana(ecotype Columbia).3.The result of RT-PCR showed that the expression of MsHppd and MsHpt induced by Nacl?PEG?ABA and low temperature especially under the condition of low temperature and ABA inducion.The expression of MsHppd and MsHpt under salt stress were increase with the salt-stress time to the peak at 24 h.The variation trendency of leaves was same as the variation trendency in stem.The variation trendency of expression under salt stress was similar with the variation trendency of expression under PEG stress but have different time reach the peak.Wefound that MsHppd expression continues to increaseed during the ABA stress and the expression is much higher than MsHpt,the distribution was more uniform in the stems and leaves.The expression under low temperature was increaseed at first and then decreased when reached the peak.There were differential expression when under different abiotic stress and the expression in leaves higher than stem.4.The determination of vitamin E content in leaves and seeds showed that low copy numbe gene inserted not have significant change in transgenic Arabidopsis thaliana leaves.There were varying degrees increased of vitamin E in leaves when the transgenetic line under salt stress(up 20.17%to 66.39%compered with wild line).Low copy number genes inserted cause a slight decrease however multi-copy gene inserted have siginificant different of vitamin E content.a-VE(19.004 mg-(100 g)-1)increased 15.89 times compared with wild plant(1.125 mg-(100 g)-1),y-VE(11.411 mg-(100 g)-1)increased 2.4 times compared with wild plant(38.845 mg-(100 g)-1),?-VE(0.511 mg-(100 g)-1)decreased 1.45 times compared with wild plant(1.254 mg·(100 g)-1).It can change the content and component distribution through overexpress the MsHppd and MsHpt.5.Determined the proline contents of Arabidopsis showed that the transgenetic lines have much higher of proline contents than wild type.Transgenetic lines have advantage when resist the salt stress compared with wild type.Texted of CAT,POD showed that the CAT and POD activity of transgenetic line was higher than wild type,the different is that the CAT activity decreased but the POD activity increase with the salt concentration increased.Chlorophyll content determination results show that the wild type under high salt stress chlorophyll a/chlorophyll b lower than transgenetic lines,showed that transgenetic lines have much higher salt tolerance under high salt stress.