蓝星睡莲花青素合成酶基因(NcANS)及启动子克隆与分析

Cloning and Analysis of Anthocyanidin Synthase Gene and Promoter in Nymphaea colorata

花青素赋予植物丰富的颜色。花青素合成酶是花青素合成途径末端的一个重要催化酶。为了研究蓝星睡莲花色形成过程中的关键酶基因NcANS的序列特征、表达规律和启动子结合元件,本研究分别以蓝星睡莲叶片和花瓣为材料,采用PCR和RT-PCR方法分别克隆了NcANS的基因序列和编码区序列,基因序列长度为1 277 bp,两个外显子中间有一个200 bp内含子;编码区序列含完整的开放读码框(ORF),为1 077 bp,编码358个氨基酸,含有DIOX-N和2-酮戊二酸-Fe2+-双加氧酶保守域,后者含有2-酮戊二酸和Fe2+的活性位点。氨基酸序列比对和系统发育分析表明,NcANS与基部被子植物无油樟ANS一致性最高,与同属侏儒卢旺达睡莲亲缘关系最近,与单子叶植物进化关系最远。荧光定量PCR结果表明NcANS的表达量在蓝星睡莲花药中最高,其次是在花瓣S3时期,在花瓣的五个发育时期呈先升高后急剧降低的趋势,在S3时期达到峰值。直接PCR技术分离得到的NcANS启动子序列长度为1 382 bp,其含有MYB、MYC等转录因子结合位点、参与响应厌氧、低温、光、茉莉酸甲酯、生长素、发育或代谢等信号的顺式作用元件。本研究结果为进一步研究NcANS基因功能和蓝星睡莲花色形成分子机理提供理论基础。

Anthocyanidins are responsible for the different colors of ornamental plants.Anthocyanidin synthase(ANS)plays an important role in catalyzing the last step of anthocyanidin biosynthesis pathway.In order to study the sequence characteristics and expression pattern of the ANS and its promoter,the gene sequence and coding region sequence of waterlily Nymphaea colorata ANS(NcANS)were cloned by PCR and RT-PCR from its leaf and petal,respectively.The length of NcANS was 1277 bp,and there was a 200 bp intron between two exons.The coding region sequence contained a complete open reading frame(ORF),1077 bp in length,encoding 358 amino acids,and included two conserved domains of DIOX-N and 2-ketoglutarate-Fe 2+-dioxygenase,the latter which involved in active sites of 2-ketoglutarate and Fe 2+.Multiple alignment and phylogenetic analysis showed that NcANS had the highest consistency with basal angiosperm Amborella trichopoda ANS in amino acid sequence,had the closest relationship with waterlily N.thermarum,and had the furthest relationship with mono-cotyledonous plant in evolution.The Real-time quantitative PCR result showed that the expression level of NcANS was the highest in anther,followed by the S3 stage of petal;the expression of NcANS in the five development stages of petal increased at first and then decreased sharply,reached the peak in the S3 stage.Additionally,the promoter sequence length of NcANS isolated by PCR was 1382 bp,which contained binding sites of transcription factors such as MYB and MYC,and cis-acting elements involved in response to signals such as anaerobic,low temperature,light,methyl jasmonate,auxin,development,and metabolism.The results of this study provide a theoretical basis for further research on the function of NcANS gene and the molecular mechanism of the formation of N.colorata.