滇水金凤C4H基因的克隆及序列分析

Cloning and Sequence Analysis of C4H Genes from Impatiens uliginosa

滇水金凤(Impatiens uliginosa Franch.)是凤仙花科凤仙花属的一年生或多年生草本植物,具有较高的观赏价值、生态价值和药用价值。C4H是花色素苷合成中的第二步重要酶,其蛋白质活性直接影响着植物中类黄酮化合物的合成,从而影响着植物花色的形成。本研究以滇水金凤的整个花器官为材料,通过提取其总RNA结合RT-PCR及RACE技术克隆出2个特异片段,其cDNA全长序列分别为1518 bp和1512 bp,编码505 aa和503 aa,分别命名为C4H1和C4H2。运用生物软件对其编码的蛋白质进行分析,发现2个蛋白均为疏水性不稳定蛋白,其三级结构存在一定差异;经系统发育树分析推测其二者为旁系基因家族。通过进一步提取其总DNA结合PCR技术获得了滇水金凤C4H1和C4H2的全长g DNA序列,分别为1689 bp和1667 bp。结果将为后续探究滇水金凤花色的形成及变异机理、凤仙花的花色改良及新品种的培育提供一定的基础数据和科学依据。

Impatiens uliginosa is an annual or perennial herb plant of Balsaminaceae with high ornamental,ecological and medicinal values.C4 H is an important enzyme in the second step of anthocyanin synthesis.Its protein activity directly affects the synthesis of flavonoids in plants,thus affecting the formation of flower colors.In this study,the whole flowers of I.uliginosa were used as experimental materials,whose total RNA was isolated by using kits.Two specific fragments of C4 H gene from I.uliginosa,were cloned by RT-PCR and RACE technologies,named C4 H1 and C4 H2,whose cDNA full-length were 1518 bp and 1512 bp,and coded 505 aa and 503 aa respectively.On the basis of protein analysis by using biosoft ware,it found that both of two proteins were unstable and hydrophobic,andthere existed some differences in the tertiary structures between them;that these two genes belonged to paralogous gene family based on analysis of their phylogenetic tree.The full-length g DNA sequences of two C4 H genes were obtained by using PCR method,which were 1689 bp and 1667 bp respectively.The abovementioned results of this study will lay a theoretical foundation and basic data for further research on the formation and variation mechanisms of flower colors of I.uliginosa Franch.flower color improvement of Impatiens and the cultivation of its new varieties.