农杆菌介导加幼苗直接形成将Bt-cry1 A(b)基因转入印度棉花(英文)

Agrobacterium-mediated Transformation in Indian Cotton (G. hirsutum) Cultivar with cry1A(b) Gene and Regeneration by Direct Shoot Organogenesis

利用农杆菌介导法用Bt cry1A(b)基因对印度栽培棉种进行非基因依赖型遗传转化和植株再生。将印度栽培棉品种Anjali(LRK 516)和LRA 5166与携带Bt cry1A(b)+nptII基因的根癌农杆菌LBA4404共培养,在含100μg·ml 1卡那霉素的筛选培养基上获得了可能的转基因直接再生苗。细菌浓度、共培养时间、感染组织的阶段和大小、标记筛选浓度、培养基成分和激素等都影响转化效果和效率。对程序进行了优化。经PCR、Southern杂交、ELISA等方法分别检测,证实了Bt cry基因的插入和表达。Southern分析表明转化植株存在3～5个基因拷贝,但CRY蛋白表达量非常低(为叶蛋白的0.003%～0.004%)且生化抗虫性很弱或基本不影响鳞翅目昆虫。尽管如此,该方案仍适用于其它CRY基因或重要经济型基因进行非基因依赖型遗传转化和再生,产生转基因棉花。

Genotype independent transformation and regeneration of Indian cotton (G. hirsutum L.) cultivars were carried out with Btcry1A (b) gene by Agrobacteriummediation. Apical meristem of elite Indian cotton cultivar Anjali (LRK516) and LRA5166 were cocultivated with Agrobacterium tumefaciens LBA 4404 carrying synthetic Btcry1A (b) + nptII genes. Putative transformants were regenerated by direct shoot organogenesis in the selection medium containing 100μg·ml1 kanamycin. Bacterial concentration, duration of cocultivation, stage and size of tissues, concentration of selection marker in the medium, media composition and growth hormones, all have influence on transformation efficiency and frequency, were optimized in our protocol. Integration and expression of the Bt cry gene was confirmed by PCR, Southern blot analysis and ELISA test respectively. Southern analysis indicated the presence of 3～5 copy of the gene number in the transformed plants. However the CRY protein expression was found to be very low (0.003%～0.004% of leaf protein) and insect bioassay shown less or no effective on Helicoverpa armegira. Nevertheless, this protocol may be used to produce genotype independent transformation and regeneration to produce transgenic cotton plants with other cry genes or any economically important genes.