【作者】 谢翔；

【导师】 赵双宜；

【作者基本信息】 山东大学 ， 遗传学， 2010， 硕士

【摘要】 植物正常发育需要适合的外界环境条件,在最适的条件下植物可以正常生长、发育,达到最大生物量和最高的生长效率；在并不适合植物生长发育的外界环境条件即非生物胁迫下,会影响植物的生长和发育,严重时可造成作物大减产甚至绝收。利用现代遗传学的方法深入研究作物响应这些非生物胁迫的机理和其应答基因,对作物进行必要的遗传改良,可以提高作物对逆境的抗性,更好地指导农业生产。小麦是世界上重要的粮食作物之一,本实验室利用不对称体细胞杂交育种新技术,成功育成了国际上首例高产、耐盐、抗旱的小麦体细胞杂交新品种——山融3号。以该新品种为研究对象,探讨其耐盐、抗旱的分子机理,寻找新的抗性基因,对今后小麦的抗逆性育种,提高植物抗逆性具有极其重要的参考价值和指导作用。李朔等利用抑制性差减杂交(suppressive subtractive hybridization, SSH)从山融3号中筛选到大量与盐胁迫相关的EST,本研究从这些EST中得到一个同时响应低温胁迫的EST序列,并利用山融3号小麦cDNA全长库,获得该EST的基因全长序列。经过序列比对获知其在Genbank中注释为wcs120,是小麦在低温春化过程中诱导表达并大量积累产物的主要基因之一。但我们分离的该基因序列有三个SNP位点,并发现其可变剪接现象。在盐、渗透以及低温胁迫下,山融3号、济南177小麦的wcs120基因具有相似的表达模式,但山融三号对胁迫的响应要更早、更强、更持久；为研究该蛋白的作用我们构建了原核表达载体,在大肠杆菌BL21中表达获得一个50 kDa蛋白,该蛋白在煮沸(100℃)后仍然保持可溶状态,与报道的结果一致；表达该蛋白质的大肠杆菌对多种逆境的抗性／耐受性大大提高,说明该基因的产物可能具有多种保护作用。为了验证其在真核生物中的功能,我们构建了GFP融合蛋白瞬时表达载体和过表达载体,转化后的拟南芥菜原生质体,GFP荧光信号广泛存在于细胞核和细胞质中。转基因拟南芥菜和烟草后代纯系,对盐、旱、低温等多种胁迫的耐受性明显提高,证实了其产物确实对植物在逆境下有保护作用。本工作为探讨植物耐逆性的机理、进一步培育耐逆作物新品种提供了有益的线索。更多还原

【Abstract】 Plant growth and development need suitable environment, under optimum conditions, it will get the maximum biomass and highest growth efficiency. Abiotic stress always cause great threat on plant growth and huge yield loss when climate is serious. So its very important to reveal the mechanism of plant resistant or tolerance to salt stress. Identify and isolate salt tolerant genes by modern genetic techniques and made some necessary genetic modification to salt sensitive plant will be greatly benefit to breeder for salt tolerance improvement of plant.Wheat is one of the important food crops in the world, to increase salt and drought tolerance of it is very crucial to its yield and quality. Shanrong No.3, the first somatic hybrid introgression line from common wheat JN177 and Agropyron elongatum, had been generated in our laboratory and with high salt and drought tolerance. To understand the mechanism of plant response to abiostress resistance, we need to study some stress-relative genes extensively, this may be very important to breeder for improve wheat abiostress resistance.Suo Li et al., obtained a large number of ESTs which related to salt stress from SR3 through the suppressive subtractive hybridization (SSH). In this paper we got a sequence from those ESTs that is greatly induced by salt stress, after clone, sequence and blast NCBI, the result shows that this gene is a cold induced protein (WCS120) gene of wheat. WCS120 is mainly induced and accumulated during the vernalization of wheat. There are some difference in wcs120 gene between Shanrong No.3 and Chinese spring, for example:3 SNPs in ORF,21 bp INDEL in promoter and there may be a alternative splicing of this gene (due to different poly A site of its mRNA). We study the expression patten of this gene in various abiostress condition (such as cold, salt and PEG) between SR3 and JN177, the result show that wcs120 gene had similar expression pattern between JN177 and SR3, while it’s expression more early, strong and durable in SR3 than JN177. The OFR of wcs120 gene were inserted into pET24a+, after transformed E.coli BL21 and induced with IPTG, we got a 50 kDa protein that is heat stable (up to 100℃), the bacteria with this protein is also cold tolerance.In order to study the function of wcs120 gene in eukaryote, we constructed two eukaryotic expression vectors, one of this is fused wcsl20 gene to green fluorescent protein (GFP), after transformation of Arabidopsis thaliana protoplast, WCS120 fusion protein is targeted to nucleus and cytoplasm; another one is wcs120 gene overexpressing vector, we transfer it to Arabidopsis thaliana (Col-0) and Nicotiana tabacum (NC89), pure lines of the transgenic plants were obtained. Transgenic plants show significant tolerance to salt, drought, cold and other stress, this result confirmed that WCS120 protein does protect plants from various abiotic stresses. This work provides some clues to investigate the mechanism of plant response to abiotic stress and must be benefit to breeder for new stress resistance varieties cultivatation.更多还原