观赏海棠McWRI1基因克隆与分析

摘要

[Objective]The plant-specific AP2 proteins,which constitute a large transcription factor family,play crucial roles in different developmental stages.AP2 proteins regulate the expressions of downstream genes through interaction with specific elements in the promoter region.[Methods]In this study,McWRI1,a AP2 transcription factor gene,was isolated from Malus crabapple by RT-PCR,and then the multiple sequence alignment and phylogenetic analyses were performed.The expression levels of McWRI1 and the wax content in different crabapple species were also detected by qRT-PCR and GC-MS.Malus prunifolia was choosed as a representative to be determined the relationship between McWRI1 and other relevant genes of wax biosynthesis,such as McWRI1,McWAX,McKCS,McKPHMT,McPKM2 and McLACS.The content of wax component was tested by GC-MS.[Results]The results demonstrated that the full-length cDNA of McWRI1 is 1 221bp,encoding 406 amino acids and the amino acids translation and sequence analysis indicated that DNA binding domain,belongs to AP2 family.Multiple sequence alignment suggested that McWRI1 protein exhibited a high sequence identity to apple MdWRI1.The gene expression of McWRI1 inhibited by low temperature and the gene exrepssions of McWAX,McKCS,McKPHMT and McLACS are declined and the contents of tricosane,nonacosane,lincleic acid,oleic acid and sixteen-eighteen alkyl acid alkyl ester are increased after low temperature treatment.The results showed that McWRI1 may participate in waxes biosynthesis in crabapple.%[目的]WRI1是一个参与调控植物种子油脂合成的转录因子,其与下游靶基因启动子区域特异的DNA序列结合,调控植物种子油脂合成相关基因的表达,但其在植物表面蜡质合成中的功能很少报道.[方法]运用PCR技术克隆McWRI1基因全长的eDNA,实时荧光定量PCR测定Mc WRI1在苹果属花红、楸子、槟子以及新疆野苹果中的表达,选择楸子作为代表,测定McWRI1与蜡质合成相关基因,如McWAX,McKCS,McKPHMT,McPKM2,McLACS表达的关系,GC-MS测定果皮蜡质成分.[结果]结果表明,McWRI1基因全长1221 bp,共编码406个氨基酸;氨基酸序列比对、系统进化树分析表明McWRI具有典型的AP2家族结构域;观赏海棠McWRI1与苹果MdWRI1的氨基酸序列一致性较高.在供试的4个苹果种果实中,花红的Mc WRI1表达量最高,新疆野苹果最低,槟子与楸子居中.以楸子为试材,设置低温处理组,可见低温处理抑制McWRI1的表达,同时下调McWAX,McKCS,McKPHMT和McLACS,并且导致二十三烷、二十九烷、亚油酸、油酸以及十六烷酸-十八烷酯的含量有明显的上升.分析认为,McWRI1可能直接和间接调控超长链脂肪酸的合成、蜡质的脂肪酸的合成以及CoA供给参与观赏海棠果实表面蜡质的合成.