灯盏花chi的克隆及其生物信息学分析

查尔酮异构酶(CHI)是调控黄酮生物合成的关键酶,分离和克隆这一酶的功能基因,对利用转基因技术进行灯盏花黄酮生物合成的调控具有重要意义。本研究采用RT-PCR和RACE技术,获得了chi cDNA全序列,GenBank登录号为GU208823.1,序列全长996 bp,开放阅读框为594 bp,编码197个氨基酸,3-Race有一个多聚腺苷酸加尾信号。应用软件预测该基因编码蛋白分子量约为21.6 kD,理论等电点为4.78。该基因编码的蛋白无跨膜结构域,其二级结构的主要构件为α-螺旋和随机卷曲。对其三级结构进行了建模,表明其结构与苜蓿chi的三级结构相似。同时根据灯盏花chi N端序列变化的特征,提出了灯盏乙素的合成可能与chi在细胞亚结构的定位及其与合成代谢相关酶形成复合酶的特异性有关。研究为利用基因工程定向改变灯盏花黄酮代谢产物奠定了基础。

Cloning of Chalcone Isomerase Gene and Bioinformation Analysis of Erigeron breviscapus (vant) Hand Mazz.

Chalcone isomerase is a key enzyme in flavone biosynthesis regulation of Erigeron breviscapus(vant) Hand Mazz.Isolation and cloning of functional genes concerning flavone biosynthesis is crucial for using gene transfer to regulate flavone metabolism in Erigeron bre-viscapus.In this paper,the chalcone isomerase gene(CHI) was cloned from Erigeron bre-viscapus via RT-PCR and RACE(rapid amplification of cDNA ends).The full-length cDNA of chalcone isomerase gene is 996 bp(GenBank Accession No.GU208823),containing a 594 bp open reading frame(ORF),encoding a 197 amino acid protein,and having a polyadenylation signal in its 3’ untranslated region.The deduced protein molecular weight of CHI was 21.6 kD and its theoretical isoelectric point was 4.78.The CHI protein did not have trans-membrane domain and was hydrophilic.Its secondary structures were predominantly constructed by α-helic and random coils were the main component.The 3D model structure of the deduced Erigeron breviscapus protein was constructed similar to the CHI protein structure from Medicago sativa.According to variation characteristics in the N-end of CHI,we supposed that the special synthesis of breviscapine(scutellarin-7-O-glucuronide and apigenin-7-O-glucuronide) in Erigeron bre-viscapus may be concerned with the position of CHI in cell sub-structure and the assembled manner of enzymes in flavone biosynthesis.This research established the foundation for di-rectional change in flavone biosynthsis in Erigeron breviscapus(vant) Hand Mazz.