花粉管通道介导的抗除草剂基因(bar)对大豆的遗传转化

SOYBEAN [GLYCINE MAX(L.) MERR.]TRANSFORMATION OF HERBICIDE RESISTANT GENE (BAR) VIA POLLEN TUBE PATHWAY

采用花粉管通道介导法将含有bar基因的pPTN140质粒DNA导入8个黑龙江省大豆主栽品种（系）,导入1384朵花,共获得1164粒种子,经2次Basta除草剂筛选后共获得3个品种的除草剂抗性植株8株.对其进行PCR以及PCR-Southern杂交检测均为阳性结果,Southern杂交结果显示,8个除草剂抗性后代植株整合数目不同,其中2株整合单拷贝基因,另外6株分别整合3～8个拷贝的bar基因.对8个抗性植株的后代进行了除草剂抗性分析,D1-D3代均出现除草剂抗性植株,表明bar基因可以在转基因植株后代中遗传,抗性遗传分析结果表明不同株系间以及株系内除草剂抗性缺乏规律性,分离比率不符合孟德尔遗传规律.说明大豆花粉管通道法转化基因存在多拷贝共抑制现象.目前获得除草剂抗性稳定的D3代株系82个.试验说明利用花粉管通道技术进行大豆转化是可行的.

Plasmid pPTN140 containing bar gene was transferred into eight soybean cultivars via pollen tube pathway, 1384 flowers were treated and 1164 seeds were harvested. Eight plants of three cultivars were survived after two times Basta screening. Positive results were given by PCR and PCR-Southern certification on the eight herbicide resistant plants. Southern blotting shown that the eight plants integrated different copies bar gene, two of them integrated single copy gene and other six plants integrated three to eight copies gene respectively. Genetic analysis was done to the offspring of the eight plants and indicated that there were still herbicide resistant plants in D1-D3 generation, which suggested that bar gene could inherit to the transformed offspring and functioned in it. Genetic analysis of herbicide resistance indicated that there was no regularity within lines or among lines, and the segregation of herbicide resistance didn＇t obey the Mendelian Law. It also indicated that there were co-inhibiting phenomena because of multiple copies gene integrated in pollen tube pathway. By now, 82 stable herbicide resistant lines of D3 generation had been obtained. The research showed that it was feasible to transform soybean via pollen tube pathway.