摘要
目的:研究目的基因在转基因植株及其后代中遗传表达的稳定性,以及目的基因表达与抗病性的关系,最终得到转基因纯合株系。方法:以采用花粉介导法将RDV运动蛋白缺陷型(RDV MP-)基因导入玉米自交系478的转基因种子(T0)作为试验材料,对其或其后代进行潮霉素抗性筛选、PCR检测、目的基因表达产物含量测定、农艺性状筛选,以及田间接种病毒的抗病鉴定。结果:通过潮霉素抗性筛选从T0种子获得了11株疑似转化植株;对T1、T2、T3代转基因植株的PCR分析证实目的基因已导入玉米植株,并显示随着转化植株世代交替,目的基因可稳定遗传给下一代,且目的基因在待测材料中的检出比例也随着代数的增加而提高;目的基因表达量的测定结果为1.83～11.57 ng/mg叶片鲜重之间;田间接种玉米矮花叶病病毒试验结果证明转化植株比对照植株的抗矮花叶病能力有了显著提高,个别株系在T1代的发病率就为0,T1、T2、T3代转化植株的抗病性逐代提高,比临近对照的抗病性提高2～5级;目的基因表达量与植株(系)的抗病性显著相关,r=0.923,P<0.01;入选纯合系的农艺性状也有较大变化,穗粒数比对照系增加约5%。结论:通过以上方法,可以筛选到转基因抗病玉米纯合株系。
Objective： To study the stability of inheritance and expression of target gene in transgenic plants and their progenies, and to reveal the relation between expression of target gene and disease resistance activities of transgenic lines, and to obtain the transgenic pure lines. Methods： The transgenic maize seeds （To） that resisted to maize dwarf mosaic virus（MDMV） were obtained by pollen-mediated approach on maize（Zea mays L.） inbred line 478, the vector carrying exogenous gene was plasmid pWM101S6 that contained RDV MP^- gene and hygromycin gene for resistant-selection. The hygromycin-screening, PCR detection, measuring expression amount of target gene, investigation of agronomic characters and desease incidence of transgenic lines were carried on. Results： 123 To seeds was selected by hygromycin-screening, it had proved that 11 plants was hygromycin-resistant plants. The results of detecting to T1 transgenic plants by PCR amplification showed that RDV MP^- gene was tranferred into the transgenic plants. The same results in detecting T2 and T3 indicated that RDV MP^- gene could stable inheritance in transgenic plants and their progeny. The expression amount of RDV MP^- gene in transgenic plants were 1.83-11.57 ng/mg fresh leaves, statistical analysis confirmed that correction existed between the expression amounts of RDV MP^- gene and disease-resistant activities of transgenic plants was highly significant（r=0.923, P〈0.01 ）. The transgenic plants were infected by MDMV on the leaf, compared with control, the level of disease resistance activities enhanced 2-5 degree. Compared with control, the agronomic characters of transgenic lines have changed largely, the Kernels per ear increased by 5%. Conclusion： Based on above results, the transgenic pure lines which were disease resistant were obtained.
出处
《生物技术通讯》
CAS
2008年第1期43-46,共4页
Letters in Biotechnology
基金
国家高技术研究发展计划项目(2001AA212131)
