Molecular cloning and characterization of six novel HMW-GS genes from Aegilops speltoides and Aegilops kotschyi
Molecular cloning and characterization of six novel HMW-GS genes from Aegilops speltoides and Aegilops kotschyi
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黑山羊草和克氏山羊草 6 个新 HMW-GS 基因的分子克隆和表征
DOI：
10.1111/pbr.12046
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发表时间：
2013-06-01
期刊：
影响因子：
2
通讯作者：
Yan, Yueming
中科院分区：
文献类型：
--
作者：
Ma, Chaoying;Yang, Yang;Yan, Yueming
利用SDS-PAGE技术，鉴定出了6个新的高分子量谷蛋白亚基（HMW-GS），分别为：ASy15*、AKx1*、AKx3*、AKx2.3、AKy20*和AKy8*。克隆完整的开放阅读框（orf），并采用等位基因特异性PCR （AS-PCR）进行测序。序列比较表明，这些新基因表现出较高的单核苷酸多态性（SNP）和InDel变异。其中AKy8*在中心重复结构域140位有一个额外的半胱氨酸残基，而AKx2.3有一个异常长的重复结构域（816 aa），这可能对面筋质量有积极的影响。系统发育分析表明，AKx1*和AKx3*属于1S基因组，AKx2.3属于1U基因组，AKy20*和AKy8*最有可能属于1S基因组。两种小麦的6个HMW-GS基因与小麦的差异发生在4.77 ~ 23.54 MYA。通过大肠杆菌的异源表达和Western blotting证实了这些内源性HMW-GS基因的真实性。
Six novel high molecular weight glutenin subunits (HMW-GS) from Aegilops speltoides (SS, 2n=2x=14) and Aegilops kotschyi (UUSS, 2n=4x=28) were identified by SDS-PAGE and designated as ASy15*, AKx1*, AKx3*, AKx2.3, AKy20* and AKy8*, respectively. Their complete open reading frames (ORFs) were cloned and sequenced by allele-specific PCR (AS-PCR). Sequence comparison demonstrated that these novel genes displayed high single-nucleotide polymorphisms (SNP) and InDel variations. In particular, AKy8* had an extra cysteine residue at position 140 in the central repetitive domain, while AKx2.3 had an unusually long repetitive domain (816 aa), which might have positive effects on gluten quality. Phylogenetic analysis showed that AKx1* and AKx3* belonged to the 1S genome, AKx2.3 to the 1U genome, and AKy20* and AKy8* most likely to the 1S genome. The divergence between the 6 HMW-GS genes from the two Aegilops species and those from Triticum species occurred 4.77-23.54 MYA. The authenticity of these isolated endogenous HMW-GS genes was confirmed through heterologous expression in Escherichia coli and Western blotting.