大豆Kunitz型胰蛋白酶抑制剂基因的分离及其在抗虫植物基因工程中的应用  

ISOLATION OF SOYBEAN KUNITZ TRYPSIN INHIBITOR GENE AND ITS APPLICATION IN PLANT INSECTRESISTANT GENETIC ENGINEERING

作　　者：高越峰[1] 朱祯[1] 肖桂芳[1] 朱玉[1] 吴茜[1] 李向辉[1] 

机构地区：[1]中国科学院遗传研究所

出　　处：《Acta Botanica Sinica》1998年第5期405-411,共7页Acta Botanica Sinica（植物学报：英文版）

基　　金：国家自然科学基金;美国"洛氏基金"资助

摘　　要：大豆Ｋｕｎｉｔｚ型胰蛋白酶抑制剂属于丝氨酸蛋白酶抑制剂，具有抗虫特性。从未成熟的大豆（ＧｌｙｃｉｎｅｍａｘＬ．）子叶中提取总ＲＮＡ，然后反转录成单链ｃＤＮＡ，以此为模板，用ＰＣＲ方法扩增出大豆Ｋｕｎｉｔｚ型胰蛋白酶抑制剂基因，并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ（＋）的ＳｍａⅠ位点上。序列分析表明：克隆片段大小为６６３ｂｐ，包含了基因完整的编码序列。编码多肽由２１７个氨基酸组成，其中包括Ｎ端２５个氨基酸组成的信号肽序列，１８１个氨基酸组成的成熟蛋白序列和Ｃ端１１个氨基酸组成的液泡定位信号序列。构建了此基因的一系列植物表达载体，用于烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．）、水稻（ＯｒｙｚａｓａｔｉｖａＬ．）、棉花（ＧｏｓｙｐｉｕｍｈｉｒｓｕｔｕｍＬ．）等作物的转化。利用棉铃虫（ＨｅｌｉｏｔｈｉｓａｒｍｉｇｅｒａＨｕｂｎｅｒ）对经ＰＣＲＳｏｕｔｈｅｒｎ杂交验证获得的转基因烟草进行了抗虫测试，结果表明，转基因烟草和对照烟草相比，具有明显的抗虫能力。Soybean Kunitz trypsin inhibitor belongs to protease inhibitor family with insecticidal properties. The soybean Kunitz trypsin inhibitor gene was obtained from the template cDNA which was synthesized from the total RNA of the isolated soybean (Glycine max L.) immature cotyledons by PCR, and cloned into SmaⅠ site of pBluescript KS(+). DNA sequence analysis indicated that the cloned 663 bp DNA fragment carried the entire coding sequence, which encoded a precursor composed of 217 amino acids, including a signal peptide composed of 25 amino acids at the N terminus, the mature protein composed of 181 amino acids and an extra 11 amino acids at the C terminus. A series of plant expression vectors have been constructed for tobacco (Nicotiana tabacum L.), rice (Oryza sativa L.) and cotton (Gossypium hirsutum L.) transformation. The transgenic tobacco was confirmed by PCR and Southern blotting. Data from bioassays of insects showed that the transgenic tobacco displayed notably resistance to the larvae of Heliothis armigera Hubner compared with wild tobacco.

关 键 词：大豆 胰蛋白酶抑制剂 抗虫 转基因烟草 基因工程 

分 类 号：S476[农业科学—农业昆虫与害虫防治] S435.72[农业科学—植物保护]