割手密醛脱氢酶ScALDH基因克隆与表达分析

发布时间：2018-02-03 10:57

本文关键词： 割手密基因克隆序列分析干旱胁迫 ScALDH　出处：《云南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：甘蔗是我国最主要的糖料作物,也是重要的能源作物,在热带和亚热带都有种植,甘蔗的生长需要一定的环境条件,特别是对水分和温度要求较为严格。从水分角度来看,干旱影响甘蔗的出苗、发根、生长速率、糖分的积累,光合速率、生理变化等等。而我国蔗区85%以上分布在无灌溉条件的旱坡地,使甘蔗的生产受到影响。割手密(Saccharum spontaneum L.)是甘蔗属内的一个野生种,由于其具有良好的抗逆性,使之成为甘蔗远缘杂交育种的重要亲本。甘蔗传统的育种采用有性杂交的方法,但随着分子生物学发展,育种学家也开始转基因技术应用于甘蔗育种中。首先找到和克隆与抗逆性有关的基因,然后利用转基因技术导入甘蔗受体细胞,使其表达,提高甘蔗的抗逆性,增加产量。本研究从甘蔗细茎野生种(割手密,Saccharum spontaneum L.)中克隆抗旱相关的基因ScALDH,并研究其在干旱处理条件下的表达情况以及分析该序列的特征。为甘蔗抗旱育种及应用奠定基础。利用RT-PCR技术克隆甘蔗的ALDH基因片段,并对其核苷酸和氨基酸序列进行分析。使用NCBI Blastx、ORF find、Mega、NCBI Conserved Domain Search等程序对其分别进行不同物种氨基酸比较、开放阅读框(ORF)寻找、进化树及保守序列分析,并用Real Time-PCR分析所克隆基因在干旱胁迫前后的表达差异。结果表明:克隆出甘蔗的ALDH基因片段,总长度为1996bp,其中蛋白质编码区(CDS)全1524bp,编码508个氨基酸。与其它物种ALDH类蛋白氨基酸序列有很高的同源性,有ALDH家族的保守序列,并含有完整的开放阅读框,系统进化树分析显示与玉米的蛋白质亲缘关系最近。Real time-PCR数据表明,在干旱胁迫下,随着干旱时间的延长,该基因的表达量呈持续积累的表达模式。总体上来说,该基因对干旱胁迫显著表达。利用RT-PCR技术克隆的甘蔗ALDH基因,属于ALDH蛋白家族的一员,具有其典型的功能域,该基因在干旱胁迫过程中参与抗旱作用。该研究结果为野生种资源开发、优良抗旱亲本选择和培育抗旱性强甘蔗品种提供了参考依据。
[Abstract]:Sugarcane is one of the most important sugar crops in China and is also an important energy crop. Sugarcane is grown in tropical and subtropical regions. The growth of sugarcane needs certain environmental conditions. From the water point of view, drought affects the emergence, root, growth rate, sugar accumulation and photosynthetic rate of sugarcane. Physiological changes and so on. And the sugarcane area in China more than 85% distributed in the dry slope without irrigation conditions. Saccharum spontaneum L.) is a wild species in the genus Sugarcane, because of its good resistance to stress. It has become an important parent of sugarcane distant cross breeding. The traditional breeding of sugarcane adopts the method of sexual hybridization, but with the development of molecular biology. Breeders also began to apply transgenic technology in sugarcane breeding. Firstly, genes related to resistance to stress were found and cloned. Then transgenic technology was introduced into sugarcane receptor cells to express them and improve the resistance of sugarcane. In this study, the drought-related gene ScALDH was cloned from wild sugarcane fine stem species (Saccharum spontaneum L.). The expression and characteristics of the sequence were studied under drought treatment, which laid a foundation for sugarcane drought resistance breeding and application. The ALDH gene fragment of sugarcane was cloned by RT-PCR technique. The nucleotide and amino acid sequences were analyzed using NCBI Blastx ORF findMega. NCBI Conserved Domain Search was used to compare the amino acids of different species, and open reading frame (ORF) was used to search for them. Phylogenetic tree and conserved sequence analysis were used to analyze the difference of expression of the cloned gene before and after drought stress by Real Time-PCR. The results showed that the ALDH gene fragment of sugarcane was cloned. The total length was 1996bp, in which the protein coding region was 1524bp, encoding 508 amino acids, which had high homology with other species ALDH protein amino acid sequences. There were conserved sequences of ALDH family and complete open reading frame. Phylogenetic tree analysis showed that protein phylogenetic relationship with maize was the most recent. Real time-PCR data indicated. Under drought stress, with the extension of drought time, the expression of the gene showed a sustained accumulation of expression pattern. The ALDH gene cloned from sugarcane by RT-PCR technique is a member of ALDH protein family and has its typical functional domain. The results provided a reference basis for the development of wild species resources, the selection of excellent drought-resistant parents and the cultivation of drought-resistant sugarcane varieties.
【学位授予单位】：云南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S566.1

【相似文献】