山定子MbFAD2基因的克隆及表达分析

Cloning and Expression Analysis of MbFAD2 Gene in Malus baccata

以山定子为试材,利用RT-PCR技术从山定子中克隆到油酸去饱和酸酶基因(MbFAD2),该基因的开放阅读框为1 149 bp,编码382个氨基酸,分子量为97.511 2 kDa,等电点为5.01,该基因被命名为MbFAD2,在GenBank中的登陆号为KC702671。二级结构分析表明,MbFAD2蛋白分子中,α-螺旋、随机卷曲和不规则卷曲分别为35.86%,16.23%,47.91%。系统进化树关系分析表明,MbFAD2与橡胶树和毛白杨的亲缘关系最近,共同形成一个分支,亲源关系最远的为中间锦鸡儿。对MbFAD2进行蛋白跨膜区分析发现,该蛋白具有6个跨膜区域。通过实时荧光定量表达研究表明:MbFAD2在山定子中的根、茎、叶和花均有表达,其中在花中的表达最高,其次是叶片。4℃低温诱导该基因在一年生山定子叶中快速表达,24 h时该基因表达量最高,48 h后该基因表达量下降;而在茎中短时间内表达不明显。结果表明:山定子MbFAD2的表达量与低温密切相关。

The cDNA sequence of MbFAD2 was cloned from Malus baccata by RT-PCR. It′s open reading frame possesses 1 149 bp,and encodes a peptide of 382 amino acids. It has a calculated molecular mass of 97. 511 2 kDa and a theoretical PI of 5 . 01 . The gene was named as MbFAD2 , and its accession nucleotide sequence number in GenBank was KC702671 . Secondary structure analysis showed that MbFAD2 protein contains 35 . 86% α-helical do-mains,16. 23% extended strand,and 47. 91% random coil. Phylogenetic tree analyses showed that MbFAD2 is clo-sest to the Populus tomentosa and Hevea brasiliensis,the farthest is Caragana korshinskii. The expression of MbFAD2 was determined by real-time quantitative RT-PCR. The result showed than the MbFAD2 was expressed in different tissue organs. The highest mRNA expression was found in flower,secondly is leaf. The MbFAD2 expression could be significantly induced by low temperature（4 ℃） for 24 h,but the stem is difference. These results suggest that Mb-FAD2 may be involved in low temperature.