柠条锦鸡儿CkLEA4-1和CkLEA6-1基因的克隆及功能分析

发布时间：2018-04-17 19:07

  本文选题：柠条 + LEA基因　； 参考：《内蒙古农业大学》2017年硕士论文

【摘要】：干旱、寒冷、盐碱等是内蒙地区常见的逆境因子,给当地作物的生长、发育及产量造成严重影响,使农业经济遭受损失,因此关于植物抗逆境胁迫的相关研究便显得尤为重要。胚胎发育晚期丰富蛋白LEA作为植物中广泛存在的一类逆境胁迫响应蛋白受到了很多研究者的关注。本研究从柠条锦鸡儿(Caragana korshinskii Kom.)中克隆得到两个LEA蛋白基因CkLEA4-1和CkLEA6-1,并围绕其胁迫相关的生物学功能展开研究,所得结果如下:1.CkLEA4-1基因的开放阅读框长为1119bp,编码373个氨基酸;CkLEA6-1基因的开放阅读框长为255bp,编码85个氨基酸。经DNAMAN软件亲疏水性分析两个LEA蛋白均为亲水蛋白。序列比对与系统进化分析表明,CkLEA4-1属于LEA_4蛋白一组,但无保守结构域,因此命名为CkLEA4-1;CkLEA6-1具有LEA_6保守结构域因此命名该基因为CkLEA6-1。2.实时荧光定量PCR检测发现CkLEA4-1的表达受干旱、脱水、高盐胁迫的诱导,而CkLEA6-1的表达则主要受冷胁迫诱导。3.甘露醇处理下,CkLEA4-1基因过表达拟南芥种子萌发率与野生型无明显差异,但子叶绿化率却高于野生型。甘露醇和NaCl胁迫处理下,CkLEA4-1基因过表达拟南芥幼苗的根长明显比野生型长,且400mM甘露醇处理下过表达株系幼苗比野生型的略大、鲜重增加。干旱胁迫处理下,生长4周的CkLEA4-1基因过表达拟南芥对干旱的耐受能力增强,MDA含量也明显低于野生型。4.冷胁迫处理下CkLEA6-1基因过表达拟南芥幼苗的根长明显比野生型长,且幼苗鲜重略高于野生型。此外在低温胁迫下,CkLEA6-1基因过表达拟南芥质膜的相对电导率在常温、4℃时无明显差别,在-20℃时过表达株系较野生型略低,过表达株系的MDA含量也低于野生型。
[Abstract]:Drought, cold, salt and alkali are common stress factors in Inner Mongolia, which have a serious impact on the growth, development and yield of local crops and cause losses to agricultural economy. Therefore, it is particularly important to study the resistance of plants to stress.LEA, a rich protein in late embryonic development, has attracted much attention from many researchers as a kind of stress response proteins widely existed in plants.In this study, Caragana korshinskii Kom.from Caragana korshinskiiTwo LEA protein genes CkLEA4-1 and CkLEA6-1 were cloned, and their biological functions related to stress were studied.The results are as follows: 1. The open reading frame length of CkLEA4-1 gene is 1119 BP, and the open reading frame length of 373 amino acid gene CkLEA6-1 gene is 255 BP, encoding 85 amino acids.The hydrophobicity of two LEA proteins was analyzed by DNAMAN software.Sequence alignment and phylogenetic analysis showed that CkLEA4-1 belongs to a group of LEA_4 proteins, but has no conserved domain. Therefore, CkLEA4-1CkLEA6-1 has LEA_6 conserved domain, so it is named as CkLEA6-1.2.Real-time fluorescence quantitative PCR analysis showed that the expression of CkLEA4-1 was induced by drought, dehydration and high salt stress, while the expression of CkLEA6-1 was mainly induced by cold stress.Under mannitol treatment, the germination rate of Arabidopsis thaliana seeds was not significantly different from that of wild type, but the greening rate of cotyledon was higher than that of wild type.Under mannitol and NaCl stress, the root length of overexpressed Arabidopsis thaliana seedlings was significantly longer than that of wild type seedlings, and the overexpressed strain seedlings under 400mM mannitol treatment were slightly larger than that of wild type seedlings, and the fresh weight of overexpressed Arabidopsis thaliana seedlings increased.Under drought stress, the overexpression of CkLEA4-1 gene in Arabidopsis thaliana was significantly lower than that in wild type.Under cold stress, the root length of Arabidopsis thaliana overexpressed by CkLEA6-1 gene was longer than that of wild type, and the fresh weight of seedlings was slightly higher than that of wild type.In addition, under low temperature stress, the relative conductivity of plasma membrane of Arabidopsis thaliana overexpressed CkLEA6-1 gene was not significantly different at room temperature (4 鈩，

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