橡胶树花青素合成调控因子HbAn1的克隆及其功能分析

Cloning and Function Analysis of R2R3-MYB Regulatory Factor for Anthocyanin Biosynthesis in Hevea brasiliensis

橡胶树转基因过程筛选效率低阻碍了转基因研究快速发展。花青素累积产生的紫红色肉眼直接可辨,是一种简单、安全的可视化筛选标记。为利用橡胶树花青素作为转基因筛选标记,本文首先以R2R3-MYB保守结构域(R2、R3)为探针调取橡胶树R2R3-MYB,并与拟南芥等其他物种花青素合成相关的R2R3-MYB进行进化分析,结果显示橡胶树scaffold0374_923317和Scaffold0598_393375与其他物种已功能验证的花青素合成调控R2R3-MYB聚到同一亚类。随后从古铜期叶片中克隆到scaffold0374_923317基因,并将其命名为HbAn1。序列分析表明,HbAn1蛋白具有R2R3-MYB蛋白的典型结构:R2R3结构域及b HLH互作结构域。定量表达分析表明HbAn1在古铜期叶片及暗培养茎秆中高水平表达,在其他时期叶片及光照培养茎秆中表达水平很低,与不同组织花青素含量正相关。最后,在烟草中组成型过表达HbAn1,使烟草叶片、花瓣、花托、花丝等组织大量累积花青素,定量分析表明其激活了这些组织后期花青素合成酶基因Nt DFR,Nt LDOX,Nt UFGT。本文首次获得橡胶树花青素累积相关的正调控因子R2R3-MYB(HbAn1),这为花青素作为橡胶树转基因可视化筛选标记提供了基因资源。

The low screening efficiency for the positive embryo of rubber trees hinders the development of transgenic research in Hevea. Red purple from anthocyanin, which can be detected by naked eyes, is a simple and safe visual marker. In order to take anthocyanin as the selective marker to improve the screening efficiency for the positive embryo of rubber trees, R2R3-MYB of rubber trees was cloned based on the conserved domain of R2R3-MYB, and the phylogenetic relationship with the anthocyanin-related R2R3-MYB of Arabidopsis thaliana and other species was analyzed, Results showed that scaffold 0374_923317 and Scaffold 0598_393375 were classified in the same sub-group with other known anthocyanin-related R2R3-MYB regulators. Then scaffold 0374_923317 isolated from the bronze leaf, and named as HbAn1, had the typical conserved R2R3 and b HLH interaction domain.Quantitative expression analysis revealed that the expression of HbAn1 decreased gradually with the development of rubber leaves, which was coincided with the anthocyanin accumulation contents of rubber leaves. The expression of HbAn1 was also coincided with green and red stems, indicating that the expression of HbAn1 was positively correlated with rubber tree anthocyanin accumulation. Finally, in constitutive overexpression HbAn1 tobacco, the anthocyanin accumulation increased extremely in tobacco leaves, petals, filaments and receptacles caused by activating the late flavonoid pathway genes Nt DFR, Nt LDOX, Nt UFGT. In this paper, for the first time, R2R3-MYB positively regulating anthocyanin accumulation in H. brasiliensis was isolated, which would provide a gene resource for the visual selection marker of H. brasiliensis transformation.