转Hpa110-42小麦分子鉴定及赤霉病抗性功能评价

摘要

[Objective] This study aims to screen Fusarium head blight (FHB) resistance plants in Hpa110-42 transgenic wheat plants and provide materials for FHB resistance breeding of wheat.[Method] T2 transgenic wheat plants were identified by PCR,Southem blot and RT-PCR,assuring whether the foreign Hpa110-42 gene was integrated and expressed in transgenic wheat plants.The resistance to FHB of transgenic wheat plants was evaluated by a dripping method to single flower,and its resistance physiology was studied.[Result] PCR,Southern blotting and RT-PCR analyses have confirmed successful integration of the foreign Hpa110-42 gene into the genome of the Yangmai 158 with 1 to 3 copies and stable inheritance and expression.Disease bioassays of transgenic plants revealed that the average percentage of diseased spikelets of transgenic lines T2-17,T2-15,T2-68,T2-44 and T2-36 were highly significantly lower than YM158.Compared with Sumai 3,the average percentage of diseased spikelets of all transgenic lines was highly significantly higher than Sumai 3 excepting line T2-17.In the transgenic lines,the average percentage of diseased spikelets of line T2-17 was significantly lower than other lines,in which T2-36,T2-11 and T2-20 have reached highly significant difference level.It is showed by physiological analysis that the activities of PAL,chitinase and β-1,3-glucanase in the high resistance transgenic plants increased faster than Yangmai 158 after inoculation.The soluble protein content in all plants had a downward trend,while its content in high resistance plants is consistently higher than others.Moreover,β-l,3-glucanase activity and soluble protein content were negatively correlated with resistance grade of FHB.[Conclusion] The foreign Hpal10-42 gene was stably inherited,normally expressed and positively participated in the regulation of FHB resistance,and Hpa110-42 transgenic wheat planns with resistance to FHB were obtained.%[目的]筛选出抗赤霉病转Hpa110-42小麦植株,为抗赤霉病小麦新品种的选育提供材料.[方法]以转Hpa110-42小麦T2植株和受体扬麦1 58为供试材料,通过PCR、Southern blotting、RT-PCR等分子技术进行外源基因的整合与表达检测,并采用单花滴注法鉴定转基因小麦的赤霉病抗性,同时探讨其抗性生理.[结果]经分子检测证明,外源Hpa110-42以1-3个拷贝整合到小麦基因组中并能够稳定遗传,在转录水平上也能正常表达.赤霉病抗性鉴定表明,株系T2-17、T2-15、T2-68、T2-44和T2-36的平均病小穗率极显著低于扬麦158.除T2-17外,其余株系的平均病小穗率极显著高于苏麦3号,均未达到苏麦3号的抗性水平.T2-17株系平均病小穗率显著低于T2-15、T2-68和T2-44,极显著低于T2-36、T2-11和T2-20株系.抗性生理分析显示,接种赤霉菌孢子后,所有植株的苯丙氨酸解氨酶(PAL)、几丁质酶、β-1,3-葡聚糖酶活性均上升,但转基因高抗植株比扬麦158上升更快,而感病株上升相对缓慢；尽管可溶性蛋白含量呈下降趋势,但转基因植株的高抗植株始终高于其它株.β-1,3-葡聚糖酶活性和可溶性蛋白含量与赤霉病抗性等级值呈极显著负相关.[结论]外源Hpa110-42整合到小麦基因组中,能稳定遗传并正常表达,正向参与了小麦赤霉病抗性调控,获得了抗赤霉病转Hpa110-42小麦植株.