滇水金凤转录因子TT8的克隆及表达分析

Cloning and Expression Analysis of Transcription Factor TT8 in Impatiens uliginosa

为探究滇水金凤(Impatiens uliginosa L.)TT8(TRANSPARENT TESTA 8)基因的功能和表达特性,并解析其对滇水金凤花色的影响,研究以滇水金凤花器官为材料,通过RT-PCR等技术克隆IuTT8基因,并对其进行生物信息学分析;利用qRT-PCR分析该基因在不同花色和不同花发育阶段的表达模式。结果表明,(1)成功克隆得到滇水金凤IuTT8基因,其编码区全长为2136 bp,编码711 aa,为亲水性不稳定蛋白,gDNA全长为3938 bp,共有6个内含子;结构域分析发现该蛋白属于bHLH超家族成员,与喜马拉雅凤仙花、山茶等物种的TT8蛋白同源且Motif基序相似。(2)IuTT8与同属植物喜马拉雅凤仙花的聚在一支,相似性约86.34%;多序列比对和系统进化分析显示TT8蛋白的结构域高度保守。(3)IuTT8基因在4种不同花色滇水金凤及其4个不同发育阶段均有表达,除白色外,其表达量均随花发育的进行呈先升后降的趋势;且IuTT8基因的表达量与花色呈正相关,其中以深红色表达量最高,白色表达量最低,深红色S3的表达量约为白色S2时期的48倍。研究表明滇水金凤IuTT8基因与花色正相关,推测其在滇水金凤花色的调控中发挥重要作用。

In order to explore the function and expression characteristics of Impatiens uliginosa TT8(TRANSPARENT TESTA 8)gene,and analyze its impact on the flower color of I.uliginosa,we cloned the IuTT8 gene by RT-PCR from I.uliginosa,and carried out its bioinformatics.qRT-PCR was used to analyze the expression patterns of the gene in different flower colors and different flower development stages of cultivars.The results showed that,(1)the IuTT8 gene of I.uliginosa was successfully cloned,and its coding region was 2136 bp,encoding 711 aa,which was a hydrophilic unstable protein.The gDNA was 3938 bp,with 6 introns.Structural domain analysis showed that the protein belonged to the bHLH superfamily and was homologous to the TT8 proteins of I.glandulifera,Camellia sinensis var.assamica and other species,and they had similar motifs.(2)IuTT8 clustered with I.glandulifera,and the similarity was about 86.34%;multi-sequence alignment and phylogenetic analysis showed that the domain of TT8 protein was highly conserved.(3)IuTT8 gene was expressed in I.uliginosa with 4 different flower colors and at 4 different stages of flower development.Except for white,the expression level of IuTT8 showed an upward trend followed by a downward trend with the progress of flower development.The expression level of IuTT8 was positively correlated with flower color,with the highest expression level in deep-red flowers and the lowest in the white-flowers of cultivars.The expression level of IuTT8 in the S3 period of deep-red flower was approximately 48 times higher than that in the S2 period of white flower.The results showed that IuTT8 gene of I.uliginosa was a transcription factor of bHLH family,which was positively correlated with flower color,suggesting that it may play an important role in the regulation of flower color in I.uliginosa.