抗草甘膦转基因油菜的抗性基因向野芥菜渗入的研究

If herbicide-resistant (HR) transgenes escape from genetically modified herbicide-resistant (GMHR) crop to their weedy relatives through pollen flow, weeds with the HR trait might produce new problems for weed control. Wild B. juncea (B. juncea var. gracilis Tsen et Lee) has become a major weed of cropping systems across western China, and extended eastward along the Yangtze River valley. The introgression of the glyphosate-resistant gene from transgenic oilseed rape to wild B. juncea may cause much serious agricultural effects. However, the study on gene flow between (GMHR) oilseed rape and wild B. juncea focused only on fitness of F1 ，F2 hybrids and BC1. The research on HR gene transmission during successive generations, chromosome behavior and genetic characteristics of successive generations, fitness and maintenance of the transgene in the natural populations are scant. .We will take the progenies (BC1F3-6, BC2F2-5, BC3F1-4) of first, second and third backcross generations (BC1, BC2 and BC3) between glyphosate-resistant (GR) transgenic oilseed rape and wild B. juncea as materials. The GR gene transmission frequency during successive four generations, the fitness of progenies in different conditions (competition and non-competition, greenhouse and field) will be assessed. Cytogenetic studies (chromosome constitution, meiotic behavior) by Flow Cytometry and genomic in situ hybridization will be conducted and evolution of chromosome number in successive generations will be analyzed. Genetic characteristics of these progenies, based on AFLP markers, will be studied. Maintenance of GR trait in field under glyphosate selection and absence of glyphosate selection pressure will be checked. Form above results, the rule of change on fitness, chromosome constitution and genetic characteristics and GR gene transmission frequency during successive four generations will be revealed, and the relationship among fitness, chromosome constitution and genetic characteristics will be detected.The process of introgression of GR transgene from GR oilseed rape to wild B. juncea and the establishment ability of progenies with GR transgene in natural condition will be verified. The long-term successive results will provide more scientific and deeper basis for safety assessment on introgression from transgenic oilseed rape to wild B. juncea.

抗除草剂转基因油菜（Brassica napus)的抗性基因如成功渗入到杂草中，将会对农田生态环境造成很大的威胁。鉴于油菜的近缘杂草野芥菜（B. juncea var. gracilis Tsen et Lee）分布广泛并有扩散趋势，且转基因油菜和野芥菜后代适合度的研究只局限于F1、F2和回交1代，缺乏对抗性基因在连续多世代杂种中的传递规律和杂种染色体组成、遗传特性、适合度的深入研究。本项目以携带抗草甘膦基因的回交1-3代自交获得的子代为对象，研究其在不同生态条件下的适合度；采用流式细胞术、基因组原位杂交技术等分析其染色体组成、减数分裂行为；采用AFLP技术检测其遗传特性；结合其在有和无草甘膦选择压下的田间持续生存能力；以及抗性基因在后代中的传递频率，探索各回交代随着自交代数的增加抗性基因的传递、染色体组成、遗传特性以及适合度的变化规律，揭示转基因油菜的抗草甘膦基因向野芥菜渗入的过程。

抗除草剂转基因油菜（Brassica napus，2n=38=AACC）的抗性基因能否成功渗入到近缘杂草中与转基因在杂交和回交后代中的持续传递能力；后代染色体的重组和遗传的稳定性以及后代在自然环境中的生存能力有关。在我国，抗除草剂转基因油菜的抗性基因能漂移到野芥菜(wild B. juncea，2n=36=AABB)中产生F1。但转基因油菜和野芥菜后代适合度的研究只局限在F1、F2和回交1代。. 本研究以抗草甘膦转基因油菜和野芥菜的正反回交1代子1代-子6代（BC1F2-BC1F7，正向和反向分别以m和p表示）、回交2代子1代-子5代(BC2F2-BC2F6)以及回交3代子-子4代(BC3F2-BC3F5)为研究对象，研究它们的出苗率、抗性基因的传递频率、适合度，以及染色体组成和遗传特性，揭示抗性基因渗入到野芥菜中的过程。. 结果表明除BC1pF2和BC1pF3的出苗率显著低于亲本野芥菜外，其余后代的出苗率在93%以上，与亲本野芥菜相当。抗性基因传递频率介于70%-100%，且随着自交代数的增加而增加。温室条件下，除BC1F2外，其余后代的总适合度与同年种植的野芥菜总适合度相当。在田间试验设计的种植密度和比例下，BC1F2-BC1F6以及BC2F2-BC2F3的总适合度都显著低于野芥菜；但BC2mF4和BC2mF5以及BC3mF3、BC3pF3、BC3mF4和BC3pF4与野芥菜相当。因此，适合度随自交和回交次数的增加呈现提高趋势。. 携带抗性基因的BC1F2-BC1F5、BC2F5和BC3F4均为混倍体；回交1代随着自交代数的增加，混倍体程度降低，表现出2n=36的稳定趋势。对回交后代染色体数为36的细胞采用BAC荧光原位杂交的方法分析染色体组成，结果表明，这些细胞的染色体组成均与野芥菜相同，为20A+16B。尽管虽然随着自交代数的增加，C-基因组特异片段每条引物被检测到的平均百分率在下降，但转基因油菜的抗性基因能渗入到野芥菜中。. 由于回交后代的适合度随着自交和回交次数的增加而呈现提高趋势，因此在防范转基因油菜的基因漂移时，除防范初始杂交，更应防范杂交后代和亲本野芥菜的再次回交。一旦抗草甘膦转基因油菜和野芥菜形成杂交1代，并与野芥菜回交形成回交后代后，就可能给农田杂草防除带来威胁。