论文部分内容阅读
【目的】克隆甘蔗的Sc CCD8,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,Gen Bank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。
【Objective】 The aim of this study was to clone Sc CCD8 of sugarcane and analyze its sequence characteristics and predict its function. The expression of ScCCD8 in different tissue parts of sugarcane, different stress treatment conditions and different growth time points was studied, and its application in genetic engineering of sugarcane Provide theoretical support. 【Method】 Based on the EST sequence of CCD8 homology fragment retrieved from NCBI, the primers were designed and the fragments of CCD8 of sugarcane were amplified. The cDNA of CCD8 was amplified from RAPD and RT-PCR using 5 ’, 3 The target fragment and the full-length cDNA sequence were predicted by bioinformatics methods. The expression of CCD8 was detected by qRT-PCR in different tissues, under different stress conditions and at different growth stages. 【Result】 The results showed that the sugarcane CCD8 was cloned and named ScCCD8, Gen Bank Accession No. KP742973.1. The full-length cDNA was 2 016 bp in length and contained 1 complete open reading frame (ORF) of 1 623 bp encoding a protein of 540 amino acids with a molecular weight of 59.534 kD. Bioinformatics analysis showed that the protein encoded by ScCCD8 is a non-secreted protein located on the chloroplast, not a typical transmembrane protein with no signal peptide sites. There are multiple glycosylation sites and phosphorylation sites and other active sites. Sequence analysis showed that ScCCD8 has high similarity with CCD8 protein of other plants. Phylogenetic tree analysis showed that the genetic relationship between sugarcane ScCCD8 and sorghum CCD8 protein was relatively close. Real-time quantitative PCR (qRT-PCR) analysis showed that the expression of ScCCD8 was tissue-specific, and its expression level in roots was the highest, which was 18 times of that in old leaves. Under the conditions of simulated severe drought stress (20% PEG), salt stress (200 mmol·L -1 NaCl), phosphorus deficiency (1/8 mmol·L -1) and nutrient deficiency (pure water culture) Under the four stress treatments, the expression of ScCCD8 in the shoot tips was induced, and the expression of ScCCD8 increased significantly after 24 h of treatment. At different growth stages, ScCCD8 was expressed in the shoot tips of sugarcane at different growth stages, and its expression level at seedling stage was higher than that at germination and tillering stages. 【Conclusion】 ScCCD8, a key gene for sugarcane strigolactone biosynthesis, was cloned from sugarcane variety ROC22 and is a member of CCD8 gene family. It is speculated that ScCCD8 may be involved in response to SLs stress in sugarcane.