甘蔗独脚金内酯生物合成基因ScCCD8的克隆与表达分析

doi:10.3864/j.issn.0578-1752.2016.14.002

摘要/Abstract

摘要： 【目的】克隆甘蔗的ScCCD8，分析其序列特征并预测其功能，研究在甘蔗不同组织部位、不同胁迫处理条件以及不同生长时间点ScCCD8的表达情况，为该基因在甘蔗基因工程育种中的应用提供理论支撑。【方法】以NCBI中检索的甘蔗CCD8同源片段EST序列为模板设计引物，扩增甘蔗CCD8的片段，采用RACE和RT-PCR技术分别从甘蔗品种新台糖22号中克隆CCD8的5′、3′目的片段和全长cDNA序列，以生物信息学方法对序列进行预测分析，利用qRT-PCR方法分析CCD8在不同组织、不同逆境胁迫条件下和不同生长时间点的表达特性。【结果】克隆获得甘蔗CCD8，命名为ScCCD8，GenBank登录号为KP742973.1。该cDNA全长2 016 bp，含有1个1 623 bp的完整开放阅读框（ORF），编码540个氨基酸，编码的蛋白质分子量为59.534 kD。生物信息学分析表明ScCCD8编码的蛋白是定位于叶绿体上的非分泌性蛋白，不是典型的跨膜蛋白，没有信号肽位点。存在着多个糖基化位点和磷酸化位点等活性位点。序列分析表明，ScCCD8与其他植物的CCD8蛋白有很高的相似性。系统进化树分析显示，甘蔗ScCCD8与高粱的CCD8蛋白亲缘关系较近。实时荧光定量PCR（qRT-PCR）分析表明，ScCCD8的表达具有组织特异性，在根中的表达量最高，是老叶中表达量的18倍。其在模拟重度干旱胁迫（20% PEG）、盐胁迫（200 mmol·L^-1 NaCl）、磷缺乏（1/8 mmol·L^-1）和营养缺乏(纯水培养)4种胁迫处理下，茎尖中的ScCCD8均被诱导表达，且在处理24 h以后，ScCCD8的表达量增加较为明显。在不同生长时期，ScCCD8在甘蔗不同生长时期的茎尖中均有表达，且在幼苗期的表达量高于萌芽期和分蘖期。【结论】从甘蔗品种ROC22中克隆获得的甘蔗独脚金内酯生物合成关键基因ScCCD8是CCD8基因家族成员，推测ScCCD8可能与甘蔗SLs响应逆境胁迫有关。

关键词: 甘蔗, ScCCD8, 独脚金内酯, 实时荧光定量PCR, 干旱胁迫

Abstract: 【Objective】The gene of Carotenoid Cleavage Dioxygenase8 (CCD8) from sugarcane (Saccharum officinarum L.) was cloned, then the sequence signature and functions were investigated, furthermore the gene expression in different tissues, different abiotic stresses and different growth times were analyzed. The objectives of the present study were to provide theoretical supports for the application of the ScCCD8 gene in sugarcane genetic engineering breeding.【Method】Using homologous cloning method to obtain the sequence of ScCCD8 gene from ROC22, reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR) were used to clone the full-length sequence of ScCCD8. The bioinformatic characteristics of the ScCCD8 was analyzed using online service. The expression profiles of ScCCD8 in various tissues, in response to different stress treatments and different growth times were investigated using quantitative real-time PCR (qRT-PCR). 【Result】CCD8 was isolated from sugarcane, named ScCCD8, which was submitted to GenBank with accession number KP742973.1. It has 2 016 bp in length containing a 1 623 bp open reading frame (ORF) and encoding 540 amino acid residues. The molecular weights of ScCCD8 encoding protein were 59.534 kD, it is not a transmembrane protein that did not contain the signal peptide sites, indicating that it is not the secretory protein. Subcellular localization prediction showed that ScCCD8 might localize in chloroplast, and it contains several active sites such as phosphorylation sites and glycosylation sites. Comparison of protein sequences similarity analysis showed that ScCCD8 had more similarity with CCD8 from different plants. Phylogenetic tree analysis showed that ScCCD8 had the closest genetic relationship with Sorghum bicolor. Expression quantity of ScCCD8 was the highest in root, which is 18 times more than in old leaves. Analysis of the expression patterns in response to abiotic stress revealed that ScCCD8 is up-regulated by PEG（20% PEG）, NaCl（200 mmol·L-1 NaCl） and phosphorus deficiency （1/8 mmol·L^-1）and nutritional deficiency（cultured in pure water） in stem tip, and the obviously increase expression was found after treated 24 hours. The expression of ScCCD8 in stem tip were various in different growth times, moreover the expression was higher in germination stage then in tillering stage.【Conclusion】Sugarcane strigolactones biosynthesis-related gene ScCCD8 was cloned from ROC22, which is the member of CCD8 gene family. It is speculated that ScCCD8 might participated in plant resistance to abiotic stresses.

Key words: sugarcane, carotenoid cleavage dioxygenase 8, strigolactones, qRT-PCR, drought stress

吴转娣，刘新龙，刘家勇，昝逢刚，赵培方，林秀琴，陈学宽，苏火生，刘洪博，吴才文. 甘蔗独脚金内酯生物合成基因ScCCD8的克隆与表达分析[J]. 中国农业科学, 2016, 49(14): 2662-2674.

WU Zhuan-di, LIU Xin-long, LIU Jia-yong, ZAN Feng-gang, ZHAO Pei-fang, LIN Xiu-qin, CHEN Xue-kuan, SU Huo-sheng, LIU Hong-bo, WU Cai-wen. Cloning and Expression Analysis of Strigolactones Biosynthesis- Related Gene ScCCD8 in Sugarcane[J]. Scientia Agricultura Sinica, 2016, 49(14): 2662-2674.

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