Cloning And Characterization Of MsHST Gene From Medicago Sativa L. And Transformation Of Arahidopsis Thaliana

In this work, Medicago saliva L. "Zhong mu No.1" has been studied via plant cloning technology, transgenic technology and phenotypic analysis to the function of gene MsHST. Compared with amino acid sequence Blast of the gene, we found that the homology between the amino acids sequence of the gene and that of as-known plants is as high as 100%, especially the sectional type of alfalfa, which indicated that the clone gene is dark urine acid method base enzyme gene of alfalfa. And we named it as MsHST. The results are showed below:1. A 1185bp and genomic DNA sequence named MsHST 393 encoding amino acidswas cloned from Medicago saliva L. "Zhong mu No.l" by PCR. MsHST gene was inserted into plasmid pB1121 with DNA recombination technology to obtain a plant expressing vector pBI- MsHST. Transgenic Arabidopsis plants were obtained by the floral dip method with Agrobacterium tumefaciens GV3101. Moreover, we found the MsHST gene has been transferred into wild-type arabidopsis thaliana genome and has been expressed by DNA, mRNA, GUS protein staining detection of transgenic plant.2. With real-time quantitative analysis of the Medicago saliva’s MsHST gene, it turned out that it had the highest amount of expression in the blade, while the minimum amount in the flower. There existed obvious differences of expression in various organs and tissues. The gene was greatly influenced by the foreign aid of hormone. With the treatment of abscisic acid and gibberellin, results of the MsHST gene’s expression were as follows:in the case of abscisic acid processing, it’s amount of relative expression gradually rised and then fell rapidly as time accumulation, reaching the peak at 10h and touching the bottom at 24h. In the case of gibberellin treatments, it showed an obvious tendency that the amount of relative expression reached the peak in 3h and the bottom in Oh.Moreover, the highest value is 6 times higher than the lowest. It was found that during different periods in the tissues of the blade, the amount of MsHST gene’s expression showed the highest in the no aging green leaves, while the lowest in the advanced aging leaves.3. With the extractions and comparisons of the chlorophyll of transgenic Arabidopsis thaliana plants and non-transgenic arabidopsis thaliana plants, it turned out that the amount of chlorophyll a, chlorophyll b, total chlorophyll in transgenic plants had increased comparing with that in non-transgenic plants. It suggests that the import of Medicago sativa gene can raise the amount of arabidopsis thaliana plants’ chlorophyll, which is closely related with the function of the MsHST gene.