甜玉米液泡转化酶2基因克隆及其等位变异分析

Cloning and Allelic Variation Analysis of Vacuolar Invertase 2 Gene in Sweet Corn

为了探究液泡转化酶2(vacuolar invertase 2,VINV2)基因在甜玉米中的等位变异情况,本研究从高糖甜玉米自交系‘B-024'和低糖甜玉米自交系‘B-029'中分离了VINV2等位基因(ZsVINV2-a和ZsVINV2-b)cDNA序列,并对其基因序列、蛋白理化性质、保守结构域与跨膜结构及同源进化关系等进行生物信息学分析。结果显示,ZsVINV2-α和ZsVINV2-b基因最大开放阅读框分别为2022和2031bp,分别编码673和676个氨基酸,且存在2处In/Del和16个SNP,其中1处In/Del存在12个碱基的插入或缺失;ZsVINV2-a和ZsVINV2-b均属于稳定的亲水性蛋白,它们的蛋白分子量分别为72975.67和73208.95Da,且ZsVINV2-a的等电点略低于ZsVINV2-b;这2个蛋白具有糖基水解酶家族32的保守结构域,均在N端有β-呋喃果糖苷酶基序和在C端有半胱氨酸催化结构域;且膜上均有1个跨膜结构域,分别位于氨基酸第41~63处和第43~65处,均属于跨膜蛋白;甜玉米ZsVINV2蛋白与普通玉米的进化关系最为相近,蛋白序列同源性为95%,其次是高粱、谷子及粟等,可见ZsVINV2蛋白的进化高度保守。研究结果将为进一步了解ZsVINV2基因的等位变异在甜玉米类糖分积累中的作用提供理论支持。

To study the allelic variation of vacuolar invertase 2(vacuolar invertase 2,VINV2)genes in sweet corn,the cDNA sequences of VINV2 alleles(ZsVINV2-a and ZsVINV2-b)were isolated from the sweet corn inbred line'B-024'with high sugar content and sweet corn inbred line'B-029'with low sugar content,and detailed bioinformatics analysis was carried out,including gene sequence,physical and chemical properties of protein,conserved domain and transmembrane structure and homologous evolution.The results showed that the maximum ORF of ZsVINV2-a and ZsVINV2-b genes were 2022 and 2031 bp,encoding 673 and 676 amino acids respectively,and there were 2 In/Del and 16 SNPs,among which 1 In/Del had 12 base insertion or deletion;Zs VINV2-a and ZsVINV2-b were stable hydrophilic proteins,and their protein molecular weights were 72975.67 and 73208.95 Da respectively,and the isoelectric point of Zs VINV2-a was slightly lower than that of Zsvinv2-b;the two proteins had conserved domain of glycosyl hydrolase family 32,both of which haveβ-fructofuranosidase motif at N terminal and cysteine catalytic domain at C terminal;both of they had a transmembrane domain on the membrane,which was located at amino acids 41~63 and 43~65,and belonged to transmembrane protein;the evolutionary relationship of Zs VINV2 protein in sweet corn was the most similar to that in common corn,and the protein sequence homology was 95%,followed by Sorghum,millet and Panicum,which showed that the evolution of Zs VINV2 protein was highly conservative.The results will provide theoretical support for further understanding the role of allelic variation of ZsVINV2 gene in sugar accumulation of sweet corn.