‘窄冠白杨1号’遗传转化体系建立与抗虫基因转化

Establishment of Genetic Transformation System for ‘Populus leucopyramidalis 1’ and Transformation of Insect Resistance Gene

‘窄冠白杨1号’(Populus leucopyramidalis 1)是一种冠形窄、耐盐碱的优良白杨派无性系。由于缺乏其遗传转化体系,因此无法通过基因工程手段对其进行遗传改良。该研究以‘窄冠白杨1号’为材料,建立了农杆菌介导的遗传转化体系,并获得了较高的遗传转化效率。首先将组培苗继代培养不同时间段(35、45、55、65 d),分别取其3种组织(叶片、叶柄、茎段)以对比分化能力的强弱,确定了继代45 d后的叶片具有最强的分化能力,其增殖倍数为8.77。随后,使用继代45 d后的叶片作为遗传转化的材料,研究不同侵染条件对转化率的影响,设立了菌液浓度、侵染时间和共培养时间各3个梯度的正交试验,对这3个因素进行最佳水平筛选,通过PCR检测和GUS染色鉴定转基因株系。结果表明‘窄冠白杨1号’遗传转化体系的最佳组合为:菌液浓度OD600=0.4,侵染时间15 min,共培养时间2 d,在该组合下转化率最高可达54.23%。最后,利用该遗传转化体系获得了转BtCry3Aa抗虫基因‘窄冠白杨1号’株系,证明该体系可以做为‘窄冠白杨1号’遗传改良的有效方法。

‘Populus leucopyramidalis 1' is an excellent poplar clone with narrow crown and saline-alkali tolerance.Due to the lack of its genetic transformation system,genetic improvement could not be achieved through genetic engineering methods.In this study,an Agrobacterium mediated genetic transformation system of ‘P.leucopyramidalis 1' was established and the high genetic transformation efficiency was achieved.Firstly,the tissue culture seedlings were subcultured at different time periods(35,45,55,65 d),and three types of tissues(leaves,petioles,and stem segments) were taken to compare the differentiation ability,and it was determined that the leaves sub-cultured after 45 d had the strongest differentiation ability,with a proliferation ratio of 8.77.Then,leaves sub-cultured after 45 d were used for genetic transformation,and the effects of different infection conditions on the transformation rate were examined.Orthogonal experiments were established with three gradients of bacterial concentration,infection time,and co-culture time to screen the best levels of these three factors,and the transgenic strains were identified through PCR detection and GUS staining.The results showed that the optimal combination for the genetic transformation system of ‘P.leucopyramidalis 1' was bacterial concentration OD600=0.4,infection-time 15 min,and co-cultivation time 2 d could reach the highest transformation rate 54.23%.Finally,the ‘P.leucopyramidalis 1' transgenic strain of BtCry3Aa insect resistant gene using this genetic transformation system was obtained,and the results proved that this system could be an effective method for genetic improvement of ‘P.leucopyramidalis 1'.