青稞B组醇溶蛋白基因5′上游调控区的TAIL-PCR克隆及序列分析

Cloning of B-hordein Gene 5′ Upstream Regulation Sequence in Highland Barley and Sequence Analysis

为了阐明青稞种子中B组醇溶蛋白合成的遗传调控机制，为分子改良大麦和小麦籽粒品质奠定基础，以青稞品种Z09和Z26的基因组DNA为模板，根据已克隆的青稞B组醇溶蛋白（B-hordein）基因的5’端序列设计三个基因特异的反向引物，分别与随机简并引物配对，进行热不对称嵌套PCR（Thermal asymmetric interlaeed PCR，TAIL-PCR）扩增，从两份西藏青稞材料中分离克隆出2个B-hordein基因的上游调控序列。序列测定结果表明，两个扩增片段长度分别为395和396bp，加上已知B-hordein基因中的GSP2引物序列与翻译起始密码子之间198bp的长度，则可得到约600bp的B-hordein基因5’上游调控序列。将所得序列与Genbank中的三个贮藏蛋白基因的对应区段进行序列比对，所得序列与来自野生智利大麦（H．chil-ense）的B3-hordein基因（Genbank登录号：AY998010）、普通小麦（T.aestivum）的低分子量麦谷蛋白亚基基因（Genbank登录号：X07747）和栽培大麦（H．vulgare）的B-hordein基因（Genbank登录号：X03103）具有81％～95％的序列相似性。推测其TATAbox位于-80bp，CAAT-likebox位于-140bp处。另外，在-300bp处存在一个胚乳盒（EndospermBox，EB），包含EM基序和GCN4基序。EM基序高度保守，GCN4基序有一个核苷酸位点的突变。此外，在约-560bp处存在一个胚乳盒类似结构。

The specific primers were designed according to the published sequences of barley B-hordein genes from highland barley Z09 and Z26. Using total DNA isolated from them as the templates, two DNA fragments of about 400 bp upstream of the known B-hordein genes was amplified by TAIL-PCR and cloned. Sequencing analysis showed the two cloned fragments contained 395 bp and 396 bp, respectively. There were 198 bp in length from GSP2 primer to translation initiation codon of B-hordein genes, so we can obtain about 600 bp upstream regulatory regions B-hordeins from highland barley .o Z09 and Z26. The two DNA fragments shared a homology ranged from 81% to 95% with the three sequences, AY998010, X07747 and X03103 published on Genbank. The putative TATA box was present at position -80 bp and CAAT-like box at position -140 bp. Besides, a putative Endosperm Box including an Endosperm Motif （EM） and a GCN4-Like Motif was found at position -300 bp. EM sequences were highly conserved in five sequences, but a base mutant presented in GCN4 sequences. Furthermore, another Endosperm-like box was found at positon -560 bp. This result establishes a base for elucidating the genetic control mechanism of B-hordein gene expression in highland barley and improving crop grain quality.