农杆菌介导的将合成的苏云金芽孢杆菌cry1Ab和cry1Ac基因转化到菜心（Brassica campestris ssp. Parachinensis）中。

Agrobacterium-mediated transformation of Brassica campestris ssp. Parachinensis with synthetic Bacillus thuringiensis cry1Ab and cry1Ac genes.

作者信息

Xiang Y, Wong W-K R, Ma M C, Wong R S C

机构信息

Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China, , , , , , CN.

Department of Entomology, University of Maryland, College Park, MD 20742-5141, USA, , , , , , US.

出版信息

Plant Cell Rep. 2000 Jan;19(3):251-256. doi: 10.1007/s002990050007.

DOI:10.1007/s002990050007

PMID:30754903

Abstract

An effective plant regeneration procedure and a gene transfer system via Agrobacterium tumefaciens were developed in Brassica campestris ssp. parachinensis. Hypocotyls from 5-day-old seedlings with 2 days pre-culture were infected with Agrobacterium strain MOG301 harboring a binary vector containing a synthetic Bacillus thuringiensis (B.t.) cry1Ab or cry1Ac gene with full codon-modification. After culture and selection on MS medium supplemented with 4.0 mg/l BAP, 2.0 mg/l NAA, 70 μM AgNO and 50 mg/l kanamycin, a number of kanamycin-resistant plantlets were regenerated. PCR and Southern blotting analysis were used to identify and characterize the transgenic plants with the integrated cry1Ab or cry1Ac gene. Western blotting analysis of the transgenic plants confirmed the expression of insecticidal proteins encoded by cry1Ab or cry1Ac. Subsequent bioassay with larvae of the Diamondback moth, Plutella xylostella, demonstrated that the transgenic plants were resistant to feeding damage.

摘要

在菜心（Brassica campestris ssp. parachinensis）中建立了有效的植株再生程序以及通过根癌农杆菌介导的基因转化系统。对5日龄幼苗的下胚轴进行2天预培养后，用携带二元载体的根癌农杆菌MOG301菌株进行侵染，该二元载体含有经全密码子修饰的合成苏云金芽孢杆菌（B.t.）cry1Ab或cry1Ac基因。在添加了4.0 mg/l 苄氨基嘌呤（BAP）、2.0 mg/l 萘乙酸（NAA）、70 μM 硝酸银（AgNO₃）和50 mg/l 卡那霉素的MS培养基上进行培养和筛选后，再生出了许多抗卡那霉素的植株。采用聚合酶链反应（PCR）和Southern杂交分析对整合有cry1Ab或cry1Ac基因的转基因植株进行鉴定和表征。对转基因植株进行的蛋白质免疫印迹（Western blotting）分析证实了cry1Ab或cry1Ac编码的杀虫蛋白的表达。随后用小菜蛾（Plutella xylostella）幼虫进行的生物测定表明，转基因植株对取食危害具有抗性。