Studies On The Transcription And Expression Levels Of Selenocysteine Methyltransferase In The Leaves Of Camellia Sinensis

Selenium is the most important vital element to Human beings. Its most important physiological function in vivo is antioxidation. Se can also improve human immune function and resistance to diseases. Se is the most effective and potential anticancer nutrient, said the American physicians. According to the rocksies, 72% of Chinese land area face the problem of deficiency of Se. To improve the level of Selenium nutrition, it is recommended to ingest the Se nutrition in the form of diet Selenium. Tea is the most popular drink in China. Drinking Se enriched tea is also a way to intake Se. Selenocysteine methyltransferase(SMT) is a key enzyme in Se metabolism in plants. It is reported that transgenic Arabidopsis thaliana plantlets with Astragalus bisulcatus SMT genes obtained the ability of accumulating high concentration of Se. But there is no records about Camellia sinensis. At present, the most common way to produce Se enriched tea is to grow tea plant on Se enriched soil. It is still a problem whether it is feasible through the way of SMT transgenic technology to increase the Se concentration in tea plant on Se enriched soil, to accumulate higher concentration of Se on soil deficient of Se. The SMT and Se concentration of the same tea plants variety but grew on soils of different Se concentrations are tested in this experiment, in order to solve the question above and the Se metabolic pathway of Se non-accumulating plants.According to the relevant complete mRNA sequence ofα-tubulin of C. sinensis (accession NO. DQ340766), the coding region ofα-tubulin gene was amplified using RT-PCR method, and its products were expressed in Escherichia coli using the pET-32a(+) vector. The construction of SMT procaryotic expression vector is done by Zhu Lin.The two fusion proteins were expressed in the form of inclusion body when induced with isopropyl-D-thiogalatopyranoside (IPTG), then was purified to immunize the rabbit to produce the polyclonal antibodys againstα-tubulin and SMT. The old and young leaves samples were collected form Shitai Se enriched soil and normal soil. The total protein extracts of the four samples was made to detect the quantity of SMT using Western blot method.At the same time, the total RNA of the four samples were extracted, and then the first strand of cDNA were synthesized using M-MLV reverse transcriptase. The specific primers of GAPDH and SMT were designed, and the most proper condition for PCR is tested, as well as the cylcles of plateau phase. The four samples'cDNA was used to do PCR, and the products were tested by the means of agarose gel electrophoresis. The grey value of the bands were tested to get the result of SMT transcription level in these four samples. In this experiment, theα-tubulin procaryotic expression vector was constructed successfully, as well as the polyclonal antibody toα-tubulin and SMT. Through the results of Western blot and semi-quantitative PCR, it is showed that the transcription and translation level of Se enriched leaves and normal leaves are the same, at the same growth stage. The SMT transcription level and the expression level of the young and the old leaves is different, the older is higher than the young. It is concluded that SMT concentration of Se enriched and normal leaves are the same. In other words, fertilizing Se will not alter the level of SMT transcription and expression level. Whether it is feasible to make tea plant accumulate more Se through the way of overexpression of SMT is not known.