基因枪介导的普通小麦共转化体系的优化  

Establishment of an Optimized Co-transformation System of Common Wheat(Triticum aestivum) Using Particle Bombardment

作　　者：贺晓岚 王建伟[1] 陈新宏 李文旭[3] 汤宏[1] HE Xiao-Lan;WANG Jian-Wei;CHEN Xin-Hong;LI Wen-Xu;TANG Hong(School of Life and Health Science,Kaili University,Kaili 556011,China;Shaanxi Key Laboratory of Genetic Engineering for Plant Breeding/College of Agronomy,Northwest A&F University,Yangling 712100,China;Institute for Wheat Research,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]凯里学院大健康学院,凯里556011 [2]西北农林科技大学农学院/陕西省植物遗传工程育种重点实验室,杨凌712100 [3]河南省农业科学院小麦研究所,郑州450002

出　　处：《农业生物技术学报》2021年第1期146-158,共13页Journal of Agricultural Biotechnology

基　　金：贵州省教育厅科技拔尖人才支持计划项目(黔教合KY字[2017]094);凯里学院博士专项基金(BS201606)。

摘　　要：高效安全的转基因技术对于推进转基因植物商业化生产、降低食品和环境安全风险具有重要意义。共转化法是获得无标记转基因植物的有效手段,为了确立筛选基因(bar)与目的基因β-葡萄糖醛酸酶(β-glucuronidase, gus)共转化小麦(Triticum aestivum)的最佳摩尔比,建立安全高效的小麦共转化体系,本研究以普通小麦‘科农199’为受体材料,完整质粒pAHC25为参照,采用基因枪介导法将Ubi-bar基因表达盒或pAHC20与Ubi-gus基因表达盒分别按摩尔比1∶1,1∶2,1∶3共6个独立共转化组合导入受体材料,经胚性愈伤组织诱导、选择培养、再生等过程筛选稳定转化子。结果表明,随着Ubi-gus摩尔浓度增加,gus基因瞬时表达率显著增加,但瞬时表达与稳定表达之间无相关性。不同组合之间转化率差异显著,其中以Ubi-bar或pAHC20∶Ubi-gus摩尔比1∶2转化率最高。Ubi-bar或p AHC20∶Ubi-gus摩尔比为1∶2时,完整质粒转化率显著低于最小表达盒,但是,摩尔比为1∶1或1∶3时,完整质粒显著高于最小表达盒的转化率。GUS染色和Southern blot检测结果表明,gus基因能在受体细胞中稳定整合、表达及遗传。除结实差,阳性株系在形态上与对照无差异。该转化体系的建立为小麦遗传转化研究打下了坚实的基础。Along with the increased commercialization and planting of a range of genetically modified(GM)crops globally, the biosafety of GM crops has become a hot topic and major public concern. To elucidate the optimal molar ratio for co-transformation and develop a safe, high-efficiency wheat(Triticum aestivum)transformation system, in this study, a combination of β-glucuronidase(gus) and a herbicide resistance gene(bar) was used to select stable transformants. Six independent co-transformation experiments were performed using either the minimal cassette Ubi-bar or the whole of pAHC20, with the Ubi-gus minimal cassette at different molar ratios. The whole pAHC25 plasmid was used as a reference, which was transferred into immature embryos of the wheat variety ’Kenong 199’. The results showed that the transient expression rate of gus gene increased significantly with the increase of Ubi-gus molar concentration, but there was no correlation between transient expression and stable expression. The transformation frequency differed significantly among combinations, in which the highest was obtained by combining Ubi-bar or pAHC20∶Ubi-gus at a molar ratio of 1∶2. The transformation frequency was significantly lower using the whole plasmid than the minimal constructs with co-transformation at a molar ratio of 1∶2, but significantly higher than that with 1∶1 and 1∶3. The stable integration, expression, and inheritance of the gus were confirmed by GUS assays and Southern blotting. The positive transgenic lines exhibited normal morphology except for poor seed set. This system may facilitate future transformation studies.

关 键 词：共转化 基因表达盒 无标记 基因枪转化 无载体骨架 小麦 β-葡萄糖醛酸酶基因(gus) bar 

分 类 号：S188[农业科学—农业基础科学]