Meng M, Gao X, Han L J, Li X Y, Wu D, Li H Z, Chen Q J
College of Agronomy, Northwest A&F University, Yangling, China.
College of Agronomy, Northwest A&F University, Yangling, China
Genet Mol Res. 2014 Jan 14;13(2):2574-89. doi: 10.4238/2014.January.14.7.
To understand the relationships between single nucleotide polymorphisms (SNPs) in the waxy gene and starch parameters in common rye, we performed sequence characterization, enzyme activity testing, amylopectin/amylose ratio evaluation, starch property testing, and correlation analysis. Specific primers were used to clone waxy from 20 rye cultivars. Sequence analysis showed that waxy was 2852 bp, including 11 exons, and sequence similarity across the 20 cultivars was over 98%. The Waxy protein showed >95% similarity with those from wheat, rice, and barley, the closest genetic relationship being with wheat Wx-A type. Waxy had multiple SNPs, most of which were located in the exons. Amino acid variants were found to be mainly distributed in the catalytic domain in an imbalanced state. Multi-factor correlation analysis revealed significant correlation among starch pasting parameters in rye flour. The Waxy protein activity was significantly negatively correlated with the amylose content and amylopectin/amylose ratio. However, pasting parameters, Waxy enzyme activity, and amylopectin/amylose content ratio were not correlated. The correlation of SNPs, the key catalytic site of Waxy, with starch parameters and enzyme activity suggested that both starch pasting parameters and Waxy protein activity were influenced by No. 260 amino acid (aa). Further, the 141 and 152 aa loci were found in the enzyme-catalyzing domain of Waxy. Interestingly, Waxy enzyme activity was also influenced by the 363 aa locus in the pliable region. These results provide important theoretical regarding the high-throughput quality identification of noodle starch, functional studies, directional selection, and molecular markers of wheat Wx subunits.
为了解黑麦蜡质基因中的单核苷酸多态性（SNP）与淀粉参数之间的关系，我们进行了序列特征分析、酶活性测试、支链淀粉/直链淀粉比例评估、淀粉特性测试和相关性分析。使用特异性引物从20个黑麦品种中克隆蜡质基因。序列分析表明，蜡质基因长度为2852 bp，包含11个外显子，20个品种间的序列相似性超过98%。蜡质蛋白与小麦、水稻和大麦的蜡质蛋白相似度>95%，与小麦Wx - A型的亲缘关系最近。蜡质基因存在多个SNP，其中大部分位于外显子中。发现氨基酸变体主要不均衡地分布在催化结构域。多因素相关性分析揭示了黑麦面粉淀粉糊化参数之间存在显著相关性。蜡质蛋白活性与直链淀粉含量和支链淀粉/直链淀粉比例显著负相关。然而，糊化参数、蜡质酶活性和支链淀粉/直链淀粉含量比例之间没有相关性。蜡质基因关键催化位点的SNP与淀粉参数和酶活性的相关性表明，淀粉糊化参数和蜡质蛋白活性均受第260位氨基酸（aa）的影响。此外，在蜡质基因的酶催化结构域中发现了第1个4 a和152 aa位点。有趣的是，蜡质酶活性还受柔韧性区域第363位氨基酸位点的影响。这些结果为面条淀粉的高通量品质鉴定、功能研究、定向选择以及小麦Wx亚基的分子标记提供了重要的理论依据。
