中间锦鸡儿CiNAC1基因的克隆与功能分析

发布时间：2018-04-19 12:22

本文选题：中间锦鸡儿 + NAC转录因子　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：植物在受到干旱、高温、盐碱、冷、虫害等胁迫以后,细胞会受到不同程度的损伤。中间锦鸡儿具有耐寒、抗旱、耐盐碱和贫瘠等特点,具有良好的防风固沙和保持水土功能。NAC转录因子家族是植物特有的、最大的转录因子家族之一,在植物应答非生物胁迫和生长发育过程中有重要的功能。本文从中间锦鸡儿中克隆得到CiNAC1基因并对其进行功能分析,具体结果如下:1.通过PCR技术,克隆得到了中间锦鸡儿CiNAC1基因1066 bp的cDNA全长序列。生物信息学分析显示,CiNAC1基因的开放阅读框ORF为921 bp,编码306个氨基酸,推导的蛋白分子量为34.57 kD,等电点为8.35,是一种亲水性蛋白,N端具有保守的NAM结构域,具有26个磷酸化位点和7个糖基化位点。2.实时荧光定量RT-PCR检测显示,中间锦鸡儿CiNAC1基因表达受干旱、高盐、脱水、高pH诱导,这表明CiNAC1基因可能与中间锦鸡儿响应逆境胁迫机制有关。3.构建了 CiNAC1-GFP融合表达载体,并转化野生型拟南芥的原生质体中,通过激光共聚焦显微镜观察其亚细胞定位,发现CiNAC1定位到细胞核中,这与它作为转录因子的功能是一致的。4.构建了 CiNAC1-HA过表达载体并转化野生型拟南芥,实时荧光定量RT-PCR检测发现获得的转基因纯合体株系中目的基因均有不同程度的表达。5.转CiNAC1基因拟南芥株系侧根数目显著多于野生型;在NaC1和甘露醇处理下,CiNAC1过表达株系种子萌发率低于野生型;乙烯处理后,过表达CiNAC1的拟南芥提前衰老。
[Abstract]:After drought, high temperature, salt alkali, cold, insect pests and other stresses, cells will be damaged to varying degrees.Caragana intermedia has the characteristics of cold tolerance, drought resistance, saline-alkali tolerance and barren. NAC transcription factor family is one of the most important transcription factors family, which has good wind and sand prevention function and soil and water conservation function.It plays an important role in plant response to abiotic stress and growth and development.CiNAC1 gene was cloned from Caragana intermedia and its function was analyzed. The results are as follows: 1.The 1066 BP cDNA sequence of CiNAC1 gene of Caragana intermedia was cloned by PCR.Bioinformatics analysis showed that the open reading frame ORF of CiNAC1 gene was 921 BP, encoding 306 amino acids, the deduced molecular weight of the protein was 34.57 KD and the isoelectric point was 8.35. It was a kind of hydrophilic protein with conserved NAM domain at its N-terminal.There are 26 phosphorylation sites and 7 glycosylation sites.Real-time fluorescence quantitative RT-PCR analysis showed that the expression of CiNAC1 gene was induced by drought, high salt, dehydration and high pH, which suggested that the CiNAC1 gene might be related to the stress response mechanism of Caragana intermedia.The fusion expression vector of CiNAC1-GFP was constructed and transformed into the protoplasts of wild type Arabidopsis thaliana. The subcellular localization of CiNAC1 was observed by confocal laser microscopy. It was found that CiNAC1 was located in the nucleus, which was consistent with its function as a transcription factor.CiNAC1-HA overexpression vector was constructed and transformed into wild type Arabidopsis thaliana. Real-time fluorescence quantitative RT-PCR analysis showed that the target genes expressed in different degree in transgenic homozygous lines.The number of lateral roots of transgenic Arabidopsis thaliana lines with CiNAC1 gene was significantly more than that of wild type, the seed germination rate of NaC1 and mannitol overexpressed lines was lower than that of wild type lines, and that of Arabidopsis thaliana with CiNAC1 overexpressed after ethylene treatment was early senescence.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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