原花青素是植物中广泛存在的一类黄酮类化合物,是人类膳食的重要营养成分,在防治病虫害方面也发挥着重要作用,花青素还原酶(ANR)是合成原花青素的关键酶。以大果球红花品种为材料,克隆得到2个CtANR基因。生物信息学分析表明,CtANR2和CtANR3的编码区分别为1 020,1 023 bp,对应基因组序列中均含有5个外显子和4个内含子,第1个外显子长度不同,其余4个外显子长度一致,内含子长度差异较大。CtANR2和CtANR3基因编码蛋白质的氨基酸数目分别为339,340个,二级结构都主要由α-螺旋和无规则卷曲构成,都属于水溶性蛋白,但CtANR2蛋白不稳定,而CtANR3为稳定的亲水性蛋白。此外,2个蛋白质都不存在信号肽和跨膜结构,可能定位于细胞外。序列比对及系统进化分析表明,CtANR2和CtANR1同源性最高,亲缘关系最近,3个CtANR蛋白与菊科植物ANR蛋白进化关系最近,与茄科、锦葵科和桑科植物ANR蛋白也同属一个大分支。组织特异性表达分析发现,CtANR2和CtANR1组织表达模式相似,都是在花中的表达量最高,初期果球表达量最低,而CtANR3则在苞片中表达量最高,根和初期果球中表达量最低。三者在不同激素胁迫下呈现出不同的表达模式,CtANR2和CtANR1在不同激素处理后表达量均下降,而CtANR3在5种激素处理后表达量均有不同程度的升高。以上结果表明,红花3个CtANR基因可能在红花不同发育阶段及抵抗非生物胁迫中有不同的分工。

As part of flavone compounds,proanthocyanidins (PAs)play important roles in defense against pests and diseases,and they are also a kind of nutritionally valuable component of human diet.Anthocyanidin reductases (ANRs)are involved in the biosynthesis of PAs.We cloned two CtANR genes from Carthamus tinctorius L..Bioinformatic analysis showed that the full-length CDS sequences of CtANR2 and CtANR3 were 1 020 and 1 023 bp,respectively.Both of the genomic sequences contained five exons and four introns,the length of the first exon was different while the other four exons were the same,and the lengths of introns varied greatly.CtANR2 and CtANR3 genes encoded proteins with amino acid number of 339 and 340,respectively,and their secondary structures were mainly composed of α-helix and random curl.Both of them were hydrophilic proteins,CtANR2 protein was unstable,while CtANR3 was a stable hydrophilic protein.In addition,the two proteins both had no signal peptide sequence and transmembrane structure,and might be located outside the cells.Sequence alignment and phylogenetic analysis showed that CtANR2 and CtANR1 had the highest homology and the closest genetic relationship.The three CtANR proteins had the closest evolutionary relationship with ANR of Asteraceae,and also belonged to the same large branch with ANR of Solanaceae,Malvacede and Moraceae.Expression analysis showed that the tissue expression patterns of CtANR2 and CtANR1 were similar with the highest expression level in flowers and the lowest expression level in early fruit balls,while CtANR3 was highly expressed in bracts and almost not expressed in roots and early fruit balls.They displayed different expression patterns under different hormone treatments.The expressions of CtANR2 and CtANR1 were decreased after different hormone treatments,while the expression levels of CtANR3 were increased to different degrees after hormone treatments.These results suggest that CtANR genes might play different roles in different development stages and resistance to abiotic stresses of Carthamus tinctorius L..