【作者】 林荆；

【导师】 李晚忱；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 水是植物生命活动的物质基础,干旱对植物有广泛的影响。在干旱胁迫条件下,植物新陈代谢发生变化,生理活动受到阻碍,生长发育各阶段受到影响,植物出现萎蔫现象。垫状卷柏(Selaginella pulvinata Maxim),俗称“九死还魂草”,极耐干旱。它能经受脱水与再水化的循环,一旦获得水分,植物枯黄卷缩的部分可以迅速张开,重新复活。有研究表明垫状卷柏这类复苏植物体内含有大量的应激代谢物—海藻糖。海藻糖对生物体有特殊保护作用,它能使许多生物在高温、脱水和冷冻等异常条件下仍保持原活性。以UDP-葡萄糖和6-磷酸葡萄糖为底物的海藻糖合成途径广泛存在于细菌、真菌和植物体内,其中由TPS1基因编码的海藻糖-6-磷酸合成酶是合成途径的关键酶。研究人员已在植物体内克隆出了TPS1基因,但除了在复苏植物体内检测到大量海藻糖积累外,在其他植物体内没有检测到海藻糖的存在。国内外已有将酵母和大肠杆菌的TPS1基因对植物体进行遗传转化研究,转化植株体内海藻糖含量有所增加,抗旱性得到增强,但植株在生长发育和形态上有不同程度的改变。探索利用耐旱能力高,适宜玉米生理代谢机制与生产要求的耐旱基因,是转基因耐旱玉米培育的技术关键。本研究选择了极耐旱的垫状卷柏做为试验材料,采用RACE技术克隆海藻糖-6-磷酸合成酶基因(SpTPS1),全长3223 bp,包含一个2790bp的完整开放阅读框(ORF),并对SpTPS1基因进行核酸序列和成熟蛋白生物信息学的初步分析。为了研究垫状卷柏海藻糖-6-磷酸合成酶基因(SpTPS1)的功能,将SpTPS1基因的开放阅读框ORF亚克隆到载体pUC119中,构建载体OpUC119。然后选择适当的酶切位点分别酶切载体OpUC119和质粒载体pRS6/AtTPS1,将ORF片段与质粒载体骨架pRS6相连,最终得到真核表达载体OpRS6。用LiAc转化法将真核表达载体OpRS6和阳性对照质粒pRS6/AtTPS1转入缺失酵母菌株tps1△,YSH290中,转化混合液涂在缺失组氨酸的半乳糖培养基上培养。结果显示野生型菌株W303-1A和缺失酵母菌株tps1△不能在培养基上生长;转化了载体OpRS6和阳性对照质粒pRS6/AtTPS1的菌株能在缺失组氨酸的半乳糖培养基上生长。将培养基中的2%半乳糖换为2%葡萄糖,挑选阳性转化子划线于该培养基中。结果显示野生型菌株W303-1A和缺失酵母菌株tps1△不能在缺失组氨酸的葡萄糖培养基上生长;转化了载体OpRS6和阳性对照质粒pRS6/AtTPS1的菌株tps1△,由于恢复了功能,故能在含有葡萄糖的培养基上生长。提取阳性菌株质粒DNA,经PCR检测正确。通过酵母功能互补试验证明了SpTPS1基因具有编码海藻糖-6-磷酸合成酶的功能。本研究从垫状卷柏中克隆了具有抗旱耐盐的SpTPS1基因,它作为培育抗旱耐盐新品种的抗旱基因,具有广泛的应用价值。并为后续的抗旱耐盐转基因新品种的培育提供了耐旱能力更高,适宜玉米生理代谢机制与生产要求的耐旱基因。更多还原

【Abstract】 Water is the fundamental material in plants, drought have a wide range of impact on plants. In drought stress conditions, plant metabolism has changed, physical activities blocked, the various stages of growth impacted, and appears wilt phenomenon.Selaginella pulvinata, commonly known as the "resurrection grass", displays extremely tolerance to desiccation. It can survive in the dehydration and re-hydration cycle, the curling yellow parts can be quickly extended and resurvived once got water. Studies have shown that the "resurrection plant", such as Selaginella pulvinata, contains a lot of stress metabolites-trehalose. Trehalose plays a special protective role in organisms, it can maintain the biological activities, even in the abnormal conditions, such as high temperature, dehydration and freeze. The trehalose synthesis pathway, which uses UDP-glucose and 6-phosphate glucose as substrate, is the most widely distributed in eubacteria, fungi and plants, the Trehalose-6-phosphate synthase, encoded by TPS1 gene, is the key enzyme in the pathway. The researchers have been cloned TPS1 in plants, but they found there is no accumulation of trehalose in vivo, except for the resurrection plants. They also have been transformed the S. cerevisiae TPS1 and E.coli otsA into plants, the positive plants increased the trehalose content in vivo and tolerance to desiccation, but had varying degrees of changes in development and morphology.It is a transgenic techno-key to gain the higher drought-resistant gene that is fit for corn physiological mechanisms and production. Our research chose the extremely drought-tolerant Selaginella pulvinata to clone trehalose-6-phosphate synthase gene (SpTPS1) by RACE, the gene whole length 3223 bp, containing 2790 bp open reading frame, and analyzed the nucleotide and mature protein through bioinformatics.To research the SpTPS1 gene function, we subcloned the ORF into vector pUC119, and then select the appropriate restriction site to respectively digest the vector OpUC119 and pRS6/AtTPS1, the ORF fragment and the pRS6 framework from pRS6/AtTPS1 were linked, and finally we got the eukaryotic expression vector OpRS6. Transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 into the host yeast strain tps1△, YSH290 by lithium acetate, and then spread the dilution onto a selection medium which contains the appropriate galactose and lacks Histidine. The result shows that wild-type strain W303-1A and deletion yeast strain tps1△can not grow in the medium; but the yeast strain which transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 can grow in the medium. We substituted 2% galactose for 2% glucose in the selection medium, and selected the positive transformants to grow on the medium. The result shows that the wild-type strain W303-1A and deletion yeast strain tps1△can not grow in the medium which contains glucose and lacks Histidine; but the yeast strain which transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 recoveried the function of deletion yeast strain tps1△, and can grow in the medium containing glucose. Extracted the plasmid DNA of the positive strains which is testified correct by PCR detection. Consequen -tly the SpTPS1 gene can be testified to encode the active trehalose-6-phosphate synthase by the yeast fuctional complementary test.In this study, we cloned drought-tolerant gene SpTPS1, it is valuable for a wide range of applications to cultivate new drought-resistant varieties. It provide a higher drought-tolerant gene that is fit for corn physiological mechanisms and production to cultivate new drought-tolerant transgenic cores.更多还原