农杆菌介导法将fad2基因的ihpRNA表达框转入甘蓝型油菜

Transferring an Inverted Repeat Expression Cassette of fad2 Gene into Brassica napus by Agrobacterium tumefaciens

为获得转基因高油酸油菜新种质,以甘蓝型油菜品种宁油12号带柄子叶为转基因受体,通过与含有油酸脱饱和酶基因(fad2)ihpRNA表达载体(pCNFIRnos)的农杆菌LBA4404共培养,将油菜fad2基因的反向重复序列表达框转入宁油12号,获得转基因植株。结果表明:4日龄的带柄子叶在含有2,4-D 1 mg/L的共培养基中预培养48 h后,再进入不定芽诱导培养基中进行培养,能显著提高不定芽的诱导频率;在含20 mg/L卡那霉素的诱导培养基中抗性绿芽的频率达到5.04%;PCR检测卡那霉素抗性苗的基因组DNA,均扩增出NPTII基因和目的基因序列表达框的特定条带。初步证明目的基因序列已经整合到了油菜的基因组中。

In order to obtain transgenic high oleic acid rapeseed germplasm,the cotyledon petioles of Brassica napus Ningyou No. 12 were selected as explant for genetic transformation and were co-cultured with Agrobacterium tumefaciens strain LBA4404 harboring the vector containing a invert-repeat expression cassettes targeting the delta-12 oleate desaturase （fad2） gene of Brassica napus. The result showed that the inductive frequency of adventitious bud was significantly increased by pre-cuhuring petiole explants of 4-day-old seedlings for 48 h in MS medium with 1 mg/L 2,4-D before sub-cultured in an inductive medium. The percentage of green adventitious bud resistant to 20 mg/L kanamycin was about 5.04%. All the transformed plants had the expected band of NPTII gene and the specific bands of target gene sequence frame by PCR analysis, which confirmed the presence of foreign DNA in the genome of transgenic plant.