| 摘要: |
| 该研究从甘蔗细茎野生种(割手密Saccharum spontaneum)中克隆抗旱相关的基因ScALDH,并分析其在干旱处理条件下的表达情况和序列特征。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 finder、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 1 996 bp,其中蛋白质编码区(CDS)全长1 524 bp,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。 |
| 关键词: 割手密, 基因克隆, 序列分析, 干旱胁迫, real time-PCR |
| DOI：10.11931/guihaia.gxzw201601013 |
| 分类号:Q943 |
| 文章编号:1000-3142(2017)03-0380-08 |
| Fund project:国家自然科学基金(31560417); 云南省应用基础研究重点项目(2015FA024); 云南省现代农业甘蔗产业技术体系建设项目(2009-2015); 云南省重点新产品开发计划项目(2012BB014); 云南省高原山地作物可持续生产系统研究创新团队项目(云科人发 [2012] 18 号)[Supported by the National Natural Science Foundation of China(31560417); the Yunnan Applied Basic Research Program(2015FA024); the Industry Technology System of Cane in Yunnan; Yunnan Provinc Key Program for New Product Development; Yunnan Plateau Mountain Sustainable Crop Production Team Innovation System Research Program( [2012] No. 18)]。 |
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| Cloning and expression analysis on the ScALDH gene of Saccharum spontaneum |
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SHEN Ming-Ming1, CHENG Zhi-Yuan1,2, CHEN Shu-Ying1,
WANG Xian-Hong1, HE Li-Lian1, LI Fu-Sheng1*
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1. College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming 650201,
China;2. Institute of Crop Sciences of CAAS, Beijing 100081, China
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| Abstract: |
| We cloned a drought resistant ALDH gene with RT-PCR gene from Saccharum spontaneum, detected the gene expression level under different drought conditions, and analyzed the nucleotide and amino acid sequences with Blastx, Conserved Domain Search, ORF finder and MEGA. We compared the amino acids among different species, and tried to find reading frames in the sequence, to search conserved Domain and to make evolutionary tree. We analyzed the gene expression level of the cloned gene in drought stress with real time-PCR. The total length of the ALDH gene fragment cloned was 1 996 bp, including 1 524 bp coding sequence, which was encoded 508 amino acids. The cloned sequence had a very high homology with other species' amino acid sequences and had the conserved sequences of ALDH family, a complete open reading frame as well. Phylogenetic tree analysis showed that protein had the closest relationship with corn. The real time-PCR data showed that the expression of the gene was expressed in a continuous accumulation mode. On the whole, the gene responds to drought stress. The cloned gene belong to the ALDH protein family, which has its typical function domain. The expression pattern analysis showed that the gene was involved in the process of drought stress. The results of this study aimed at developing the excellent resources of wild species, as well as to explore the drought resistance of these resources. The study provides the basis for the selection of the parents of the fine and drought resistance in the future. |
| Key words: Saccharum spontaneum, gene cloning, sequence analysis, drought stress, real Time-PCR |
