青稞HvBAS1基因的克隆及表达分析

Cloning and Expression Analysis of HvBAS1 in Hordeum vulgare L. var. nudum Hook. f.

细胞色素P450单加氧酶(CYP450)是参与植物代谢的最大酶家族,其中CYP734A亚家族成员广泛参与植物激素油菜素类固醇(BRs)的失活。该研究以青稞农家品种‘肚里黄’幼苗为实验材料,通过人工合成激素24-表油菜素内酯(24-eBL)和BRs合成抑制剂油菜素唑(BRZ)处理,分析BRs对青藏高原特色作物青稞(Hordeum vulgare L. var.nudum Hook. f.)的影响;采用RT-PCR技术从青稞中克隆HvBAS1基因,并运用实时定量PCR检测其表达特征,为深入分析HvBAS1基因的功能奠定研究基础。结果显示:(1)24-eBL能够显著促进青稞幼苗的生长,而BRZ处理后幼苗长势明显减缓。(2)从青稞中成功克隆到2个与拟南芥BRs失活基因BAS1高度同源的CYP734A亚家族基因,即HvBAS1-1和HvBAS1-2;HvBAS1-1开放阅读框全长1 629 bp,编码542个氨基酸;HvBAS1-2长1 689 bp,编码562个氨基酸,二者氨基酸序列相似性为76.42%;亚细胞定位预测结果显示二者均存在于内质网。(3)实时定量PCR检测发现,青稞HvBAS1-1与HvBAS1-2的表达模式完全不同,其中HvBAS1-1在青稞根中的表达量高于叶中,而HvBAS1-2在叶中的表达量高于根,且随着苗龄的增大,HvBAS1-1在根中的表达呈先升高后降低的趋势,HvBAS1-2在叶片中呈先降低后升高的变化趋势;BRZ处理青稞幼苗后,其HvBAS1-1与HvBAS1-2基因的表达较对照均显著下调,且HvBAS1-2下调更为明显。研究表明,HvBAS1-1和HvBAS1-2很可能都参与了青稞内源BRs的失活,但二者的功能存在差异。

Cytochrome P450 monooxygenase(CYP450) is the largest enzyme family involved in plant metabolism, among which CYP734 A subfamily members are widely involved in the inactivation of plant hormone Brassinosteroids(BRs). In order to analyze the effects of BRs on Hordeum vulgare L. var. nudum Hook. f.(highland barley), which is a characteristic plant in Qinghai-Tibet plateau, we treated the seedlings of highland barley cultivar ‘Dulihuang’ with the synthetic hormone 24-epibrassinolide(24-eBL) or BRs synthetic inhibitor brassinazole(BRZ). In order to deeply analyze the function of HvBAS1, we cloned the cDNAs from highland barley by RT-PCR, and detected their expression characteristics by real-time quantitative PCR. The results showed that:(1) 24-eBL could significantly promote the growth of highland barley seedlings, while BRZ was opposite.(2) Two subfamily members of CYP734 As were cloned from highland barley. Due to their high homology with Arabidopsis BAS1, they were named HvBAS1-1 and HvBAS1-2. HvBAS1-1 was 1 629 bp in length and encoded 542 aa, while HvBAS1-2 was 1 689 bp in length and encoded 562 aa. The homology of amino acid sequences between them was 76.42%. The results of subcellular localization predicted that both of them existed in endoplasmic reticulum.(3) Real-time quantitative PCR detection showed that the expression patterns of HvBAS1-1 and HvBAS1-2 were completely different, in which the expression of HvBAS1-1 in the roots was higher than that in the leaves, while the expression of HvBAS1-2 in the leaves was higher than that in the roots. With the increase of seedling age, the expression of HvBAS1-1 in the roots showed a trend of first increasing and then decreasing, but HvBAS1-2 decreased firstly and then increased in leaves. After BRZ treatment, both HvBAS1-1 and HvBAS1-2 were significantly down-regulated compared with the control, and the expression level of HvBAS1-2 were decreased even more. The results indicated that both HvBAS1-1 and HvBAS1-2 were probably involved in inactivation of endogenous BRs in highland barley, but their functions were different.