为明确与烟草低温早花有关的调控基因，利用生物信息学方法结合RT-PCR和SMART RACE技术，从烟草中克隆了2个E3泛素蛋白连接酶HOS1基因cDNA全长序列，分别命名为NtHOS1a（GenBank登录号：KU558689）和NtHOS1b（GenBank登录号：KU558690）。NtHOS1a基因全长为3599 bp，编码963个氨基酸；NtHOS1b基因全长为3578 bp，编码954个氨基酸；两基因氨基酸序列的一致性为95%，与拟南芥HOS1蛋白（NP_181511）的序列一致性分别为51%和50%。两基因编码蛋白的N末端均含有一个E3泛素连接酶特征的环指结构域。荧光定量PCR分析表明：在正常生长条件下，NtHOS1a和NtHOS1b在烟草根、茎、叶中表达强烈，12℃低温处理10 d后两基因在烟草根、茎、叶中的表达量均有不同程度的降低，说明在烟草根、茎、叶组织中NtHOS1a和NtHOS1b的表达量受低温胁迫的负向调控。%To identify the genes related to the regulation of early flowering at low temperature in tobacco, two full-length cDNAs encoding different E3 ubiquitin ligase genes (NtHOS1a and NtHOS1b) were cloned from Nicotiana tabacum by bioinformatics, RT-PCR and SMART RACE technologies. GenBank accession numbers of NtHOS1a and NtHOS1b were KU558689 and KU558690, respectively. The putative full-length cDNA sequences of NtHOS1a and NtHOS1b were 3 599 bp and 3 578 bp, and these two genes encoded the predicted polypeptide of 963 and 954 amino acids, respectively. Amino acids identity between NtHOS1a and NtHOS1b was 95%. Putative polypeptide in the two genes matched Arabidopsis thaliana HOS1 (NP_181511) polypeptide sequence with the identity of 51% and 50%,respectively. The two proteins shared highly conserved N-terminal RING domains of E3 ubiquitin ligases. qRT-PCR showed that under normal growing condition, NtHOS1a and NtHOS1b transcripts accumulated to a high level in root, stem and leaves. After 10 days of 12 ℃ treatment, the expression levels of the two genes were decreased to different degrees, this suggested that NtHOS1a and NtHOS1b expressions in tobacco root, stem and leaves were negatively regulated by low temperature.
展开▼
