【作者】 刘娜；

【导师】 郭三堆； 罗淑萍；

【作者基本信息】 新疆农业大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 植物中存在着控制和应答氧水平的敏感体系,从而维持稳定的氧水平,这对于避免不必要的能量丧失以及植物稳定生长至关重要。已知透明颤菌血红蛋白基因(vgb)在大肠杆菌中的表达明显促进了细胞在微氧条件下的生长和蛋白质合成能力。因此本文对转透明颤菌血红蛋白基因(vgb)的抗虫棉进行研究,通过该基因对棉花代谢水平和棉花生长的影响,确定其在抗虫棉增产和抗涝方面的功能和作用。首先利用分子生物学方法对转vgb基因的抗虫棉进行鉴定:PCR检测到Bt基因和vgb基因已整合到棉花基因组中;Southern杂交确定vgb基因在抗虫棉中的拷贝数为多个拷贝;用Bt免疫试纸条检测转vgb基因的抗虫棉有Bt杀虫蛋白存在,并进一步用ELISA检测Bt蛋白表达量;并用荧光定量PCR技术检测了vgb基因在棉花中的表达量。表明vgb基因和Bt基因均整合到棉花基因组中,且稳定的表达。通过大田试验对转vgb基因抗虫棉的三个株系进行棉花生理活动跟踪调查得出以下结论:转vgb基因抗虫棉在表现对棉铃虫显著抗性的同时,棉花种子的子叶展开和真叶出现从总体上显著早于对照棉花材料;叶绿素含量较对照提高15%-20%;棉花花期较对照提前3-5天;单株结铃数较对照增多,但衣分没有变化;三个株系的棉花产量均明显提高,其中转vgb基因株系1的棉花子棉较单转Bt的棉花增加了134.5%,皮棉较其增长了150.2%,表现最优;生育期较对照缩短了10天左右;对转vgb基因的抗虫棉进行苗期和花期为期15天的淹水试验,检测SOD、CAT、MAD、PRO等多项生理指标,并对淹水后的棉花进行农艺性状和产量性状的调查,证明转vgb基因的棉花较对照抗涝性明显增强。本研究首次报道了透明颤菌血红蛋白基因在棉花的增产和抗涝性提高两方面具有作用,并注意到了叶绿素含量测定可作为检测vgb基因表达的指标。为vgb基因在棉花里表达所产生的生长增加和代谢物变化情况提供数据,并为推进转vgb基因高产抗虫棉的产业化应用和其它转vgb基因农作物或蔬菜研究提供依据。更多还原

【Abstract】 There is a sensitive system controlling and responding to the level of oxygen in the plants. Thus, it is important to maintain a steady level oxygen to avoid the unnecessary loss energy and to ensure survival. A novel genetic response to enhance oxygen metabolism has been observed in the microbial world: The obligate aerobic, Gram-negative bacterium Vitreoscilla synthesizes elevated quantities of a novel homodimeric hemoglobin (VHb) in oxygen-limited cultivations. Expression of VHb in Escherichia coli increases specific growth rate, final cell density, and cloned reporter enzyme expression in oxygen-limited cultures. This study had developed vgb (Vitreoscilla hemoglobin gene) transgenic cotton,we studied the influence to plant metabolic level and growth.This work showed the function both yield improvement and water logging resistance.of vgb gene in insect-resistant cotton.In this study we proved that vgb gene in insect-resistant cotton. containing and expressing the vgb gene and Bt gene respectively, PCR and Southern blot analysis confirmed their transgenic status: PCR result was that there was no specific band appeared in negative control, but a specific band similar to positive control was amplified from the test sample; Southern blot result was there two vgb-specific hybridizing bands. At the same time we found that there were more vgb insecticidal protein at seedling stage than at summer stage of cotton life. Finally, we tested that there were Bt insect-resistant proteins of different levels in both bivalent insect-resistant cotton leaf and transgenic Bt pest-resistant cotton leaf by Bt-Cry1Ab/1Ac rapid paper strips test method. Fluorescence real-time quantitative PCR, the relative expression level of vgb gene in a different transgenic cotton Lane is detected, and had difference expression.The transgenic cotton plants had showed obvious cotton bollworm resistance. Simultaneously, we also found their geminating speeded up, leave chlorophyll content improved by 15-20%, flowering date moved up 3-5 day ,the boll number increased significantly,but the seed-cotton weight per-boll is no change . The yield increased significantly, thereinto ,the transgenic cotton 1 lint weight seed increased 134.5%, cotton yield increased 150.2%.Carry on the inundation experiment during seedling and flowering period, Observe Cotton yield and its yield components were affected significantly by different cotton Species. Examine several physiology index signs of SOD, CAT, MAD, PRO etc,provrd vgb Gene in Transgenic Insect-resistant Cotton have water logging resistance.This work had proved that the transgenic cotton expressing Vitreoscilla hemoglobin showed both yield improvement and water logging resistance. We also noticed that chlorophyll content was a good mark to judge the expression of Vitreoscilla hemoglobin in transgenic plant. This work had laid a research foundation for developing yield improvement transgenic crops in further.We manage to reach the goal of increasing the yield of transgenic plants and cultivate new plant vatities with pest-resistance by applying transgenic approaches, and eventually in the hope of cultivating new varities of crops with both highly-steady-yield and pest-resistance. will undoubtedly promote the studies and the commercialization of the GMO crops.更多还原