摘要
通过根癌农杆菌的介导将慈姑蛋白酶抑制剂基因（ＡＰＩ）导入小白菜（Ｂｒａｓｓｉｃａ ｃａｍ ｐｅｓｔｒｉｓｓｓｐ．ｃｈｉｎｅｎｓｉｓＬ．）。Ｋａｎａｍ ｙｃｉｎ 筛选浓度为１０～１５ ｍ ｇ·Ｌ－ １，抑制农杆菌生长的抗生素选用Ｃａｒｂｅｎｉｃｉｌｌｉｎ，浓度为３００～５００ ｍ ｇ·Ｌ－ １。小白菜带柄子叶被农杆菌感染后，抗性芽的诱导频率为０．２％ ～２．０％ 。加入适量的ＡｇＮＯ３ 不仅可以抑制过度生长的农杆菌，还可以使抗性芽的诱导频率约提高到５％ 。试验获得转基因植株９７ 株。其中８１ 株ＰＣＲ 检测为阳性。部分转基因植株经ＰＣＲＳｏｕｔｈｅｒｎ 及ＲＴＰＣＲ 的进一步分子检测分析，证明ＡＰＩ基因已被整合进小白菜植株的基因组中。转基因小白菜的叶片离体饲虫初步试验结果表明：对鳞翅目害虫菜青虫的生长发育有一定的抑制作用，其幼虫摄食转基因植株叶片后，平均体重、体长均分别比对照（摄食非转基因植株叶片）减轻和短小，且约有２７．８％ 的幼虫不能正常羽化，而对照全部正常羽化。
The arrowhead proteinase inhibitor(API)gene was introduced into sm all Chinese cabbage (B. chinensis) via Agrobacterium tum efaciens m ediated transform ation. Cotyledon explants with petioles w ere co cultivated w ith Agrobacterium strain LBA 4404 carring pBI APIplasm id.The pBI APIcontained aneom ycin phosphotransferaseⅡgenefor Kanam ycin resistance.Following co cultivation,the explantsweretransferred to fresh shoot induction m edium with 10～15m g·L- 1 Kanamycin and 300～500m g·L- 1 carbenicillin.The transform ation ratesw ereabove0.2% ～2.0% .In thepresence ofAgNO3 in them edium ,the transform ation rates were up to 5% . Transgenic plants were initially identified by Kanam ycin,and subsequently the APIgene wasconfirm ed by PCR Southern analysis.Insect biossay dem onstrated thattheaccumulation ofAPIin transgenicplantscould reducedam age by Pierisrapaelarvaeand inhibitlarvaldevelopm ent.
出处
《上海农业学报》
CSCD
1999年第4期4-9,共6页
Acta Agriculturae Shanghai
基金
上海市农业委员会科技兴农重点攻关项目
关键词
小白菜
慈姑蛋白酶
抑制剂
抗虫性
转基因植株
Sm allChinese cabbage
Cotyledon with petiole
Agrobacterium tum efaciens m ediated transform ation
Arrow head proteinaseinhibitor(API)
Insect resistance
Transgenic plants
