【作者】 赵小强；

【导师】 肖兴国；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 小麦(Triticum aestivum L.)是世界上最重要的粮食作物之一,也是喜氮作物,其产量和/或品质往往与氮(特别是硝态氮,NO3-)的施用量呈一定程度的正相关。本研究旨在探索是否可以通过导入外源的硝酸还原酶基因(Nitrate Reductase Gene, NR)来提高小麦对NO3-的利用效率,从而在保持其产量和/或品质的同时减少氮肥的施用量,或者在不提高氮肥施用量的条件下提高其产量和/或品质,从而减少氮对环境的影响。本研究以两个冬性小麦品种“农大146”(ND146)和“冀麦6358”(JM6358)的幼胚愈伤为外植体,通过农杆菌介导法导入烟草NR基因,获得了转基因的小麦植株；对转基因T1代植株的硝酸还原酶活性、硝酸盐含量与T2代种子的千粒重和蛋白质含量进行了测定,获得如下主要结果。1PCR检测表明,ND146和JM6358的转化率分别为1.68%和0.4%。转基因植株T0和T1代的PCR-Southern和Southern blot检测显示,NR基因不仅整合到小麦基因组DNA,而且能够传递到下一代。2不论在KNO3诱导前还是诱导之后,“转基因农大146"(NR-ND146)的T1代均有71.4%的植株的硝酸还原酶活力(NRA)明显高于对照；在“转基因冀麦6358"(NR-JM6358)的T1代植株中,诱导前只有6358-17-5的NRA低于对照,而来源于其它5个T0代株系的NRA经50mM的KN03诱导后活力均高于对照。3在检测的15株NR-ND146和17株NR-JM6358的T1代植株中,硝酸盐含量(NC)的变化范围分别是2.8-44.8μg/g FW和4.5-29.1μg/gFW。与野生型对照相比,NR-ND146有8株NC明显降低,而NR-JM6358则有13株。4在T1代叶片被采样的植株中,NR-ND146所有植株的T2代种子千粒重均显著高于对照,而NR-JM6358的T2代种子千粒重明显增加的植株只占50%,但两者的T2代种子的蛋白含量都明显高于对照。5在T1代叶片没有被采样的植株中,70.8%的NR-ND146和65.4%的NR-JM6358的T2代种子千粒重显著地高于对照；前者种子蛋白质含量提高的植株占70.8%,而后者只占50%。上述结果表明,在不增加施氮量的情况下,组成型表达外源硝酸还原酶基因可以显著改善小麦对氮的利用效率,从而提高籽粒的蛋白质含量和千粒重。更多还原

【Abstract】 The wheat (Triticum aestivum L.), typical nitrogen (N)-preferring plant, is one of the most important cereals in the world. Its yield and/or quality are some extent positively correlated with the application rate of nitrogen compounds (especially nitrate, NO3-). The purpose of the present work was to test whether or not introduction and expression of a foreign NR gene in wheat could increase N use efficiency and hence improve quality and/or yield without augmenting N supply, or could maintain quality and/or yield with a diminished use of N fertilizer.A tobacco NR gene under the control of CaMV35S promoter was transferred to wheat embryogenic calli derived from immature embryos of two winter cultivars,"Nongda146(ND146) and "Jimai6358"(JM6358), and the transgenic plants ("NR-ND146" and "NR-JM146") were regenerated. The nitrate reductase activity (NRA) and nitrate content (NC) of Ti plants,1000-grain weigh and protein content (SPC) of T2seeds, were analyzed. The main results were listed as follows.1. The transformation frequency were1.68%and0.4%for "ND146" and "JM6358", respectively, by PCR assay. The foreign NR gene was successfully integrated into wheat genome and passed on to the offspring, revealed by PCR-Southern and Southern blot assay in the To and T1generation.2. Compared with WT, the T1offspring of NR-ND146had a significant higher NRA in71.4%individuals tested, no matter with or without KNO3inducement. However, in NR-JM146descendants, all tested Ti individuals from5To line displayed remarkably stronger NRA than WT when induced with KNO3. Without the induction, one individual (plant6358-17-5) even showed lower NRA than WT.3. In NR-ND146family, the foliar NC of15Ti individuals varied from2.8to44.8μg g-1FW, and8plants displayed significantly lower NC than WT. In the family of NR-JM146, the foliar NC of17Ti descendents also varied from4.5to29.1μg g-1FW, and13T1individuals had the NC remarkably lower than WT.4. In all leaf-sampled Ti plants of NR-ND146family, the T2seeds had a very significant higher1000-grain weight than WT, whereas in the NR-JM6358family, only50%of leaf-sampled T1individuals displayed such a significant increment. All leaf-sampled Ti plants in both NR-ND146and NR-JM6358families had the SPC much higher than WT.5. In leaf-intact Ti plants,70.8%and65.4%of individuals in NR-ND146and NR-JM146family had the grain remarkably weighter than WT, respectively. Overall70.8%T1plants increased their SPC of NR-ND146family, but only50%T1plants increased their SPC of NR-JM6358family.These results indicated that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.更多还原