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目的克隆宁夏枸杞类黄酮/异黄酮生物合成途径中的关键酶查耳酮合成酶(CHS,EC2.3.1.74)。方法以宁夏枸杞为材料,利用同源克隆和RT-PCR技术得到1条CHS基因,并对其进行生物信息学分析。结果该家族的cDNA片段长为1 148 bp,编码382个氨基酸,蛋白相对分子质量为4.168×104,等电点为6.22。氨基酸序列和结构分析显示CHS基因家族有一个活性位点,即查耳酮和二苯乙烯合成酶活性位点。宁夏枸杞CHS(LyCHS)蛋白很可能定位在细胞质。对二级结构和三级结构进行预测,发现LyCHS蛋白中无规则卷曲126个,α螺旋和β折叠分别为166和25个,延伸链65个。系统进化分析表明,LyCHS基因与马铃薯野生种、马铃薯、番茄具有很高的同源性。该基因已在GenBank上注册,基因序列登录号为JQ964237。结论首次克隆了LyCHS基因片段,为研究其表达特性以及功能提供了基础。
Objective To clone the chalcone synthase (CHS, EC2.3.1.74), a key enzyme in flavonoid / isoflavone biosynthesis pathway of Ningxia wolfberry. Methods Ningxia wolfberry as material, using homologous cloning and RT-PCR technology to get a CHS gene, and its bioinformatics analysis. Results The cDNA fragment of this gene was 1 148 bp in length and encoded a polypeptide of 382 amino acids with a relative molecular weight of 4.168 × 104 and an isoelectric point of 6.22. Amino acid sequence and structural analysis revealed that the CHS gene family has an active site, namely chalcones and stilbene synthase active sites. Ningxia wolfberry CHS (LyCHS) protein is likely located in the cytoplasm. By predicting the secondary structure and tertiary structure, we found that there are 126 random coils in LychS protein, 166 and 25 α-helices and β-sheets and 65 extension chains respectively. Phylogenetic analysis showed that LyCHS gene has high homology with wild potato, potato and tomato. This gene has been registered in GenBank and the gene sequence accession number is JQ964237. Conclusion The LyCHS gene fragment was cloned for the first time, which provided a basis for studying its expression characteristics and function.