二溝黃芪富硒基因smt1轉(zhuǎn)化紫花苜蓿的初步研究.pdf

1、 分類號(hào) Q78 學(xué)校代碼 10129 U D C 557 學(xué) 號(hào) 09210013 二溝黃芪富硒基因 smt1 轉(zhuǎn)化紫花苜蓿的初步研究 Primary study on

2、Medicgo Sativa transformation of the Selenium-hyperaccumulation Gene smt1 from Astragalus bisulatus 申 請(qǐng) 人：李 晶 學(xué)科門類：理 學(xué) 學(xué)科專業(yè)：遺傳學(xué) 研究方向：植物抗逆分子生物學(xué)與基因工程 指導(dǎo)教師：王茅雁 教 授 任衛(wèi)波 副研究員 論文提交日期：二〇一二年五月 Primary study on Medicgo Sa

3、tiva transformation of the Selenium-hyperaccumulation Gene smt1 from Astragalus bisulatus Abstract Selenocysteine methyltransferase (SMT) specifically methylates selenocysteine to produce methylselenocystein which is a

4、non-protein amino acid and accumulates in plant cell, therefore the content of selenium in plant increased. In this paper, the regeneration technology of alfalfa in vitro was optimized, and smt1, an important gene for se

5、lenium hyperaccumulation in Astragalus bisulcatus, was cloned from this plant. Then the smt1 gene was introduced into alfalfa by Agrobacterium-mediated transformation so as to improve the selenium-accumulating ability of

6、 the transgenic plants. The main results are as follows: (1)The suitable medium to induce callus from alfalfa was SH medium +2.0mg/L 2,4-D+ 0.25mg/L KT+ 0.25mg/L TDZ+7.5g/L agar +30g/L sucrose. (2)The suitable medium to

7、induce adventitious buds from alfalfa was MSO medium+2.0mg/L 2,4-D+0.5mg/L 6-BA+2g/L casein hydrolyzate+7.5g/L agar+30g/L sucrose. (3)The suitable explants were cotyledon and hypocotyl from 5-day old seedlings of alf

8、alfa. (4)The coding region cDNA of Astragalus bisulatus smt1 was synthesized artificialy. It contains 1017 base pairs and encodes a polypeptide composed of 338 amino acid residues, with a molecular weight of 36.7 kD and

9、an isoelecric point of 4.69. (5)The cDNA fragment of smt1 was inserted into plant expression vector pCAMBIA1301, and then the recombinant plasmid was introduced into the strain LBA4404 of Agrobacterium tumefaciens. (6)Th

10、e Agrobacterium-mediated transformation of cotyledons of alfalfa was performed, and 15 regenerated plants with resistance to hygromycin were obtained by screening and regeneration culture. (7)Six transgenic plants were c