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枸杞查耳酮异构酶LcCHI1基因的克隆与表达分析
乔枫
0
(青海师范大学 青藏高原药用动植物资源重点实验室/高原科学与可持续发展研究院,西宁 810008)
摘要:
为探究枸杞查耳酮异构酶CHI1基因在不同组织中的表达模式和功能,利用同源克隆的方法获得1个枸杞(Lycium chinenseLcCHI1基因(Genbank No. OR026273),通过生物信息学方法分析其理化性质、结构及进化关系,利用荧光定量PCR(qRT-PCR)技术对其组织表达的特异性进行分析,并进行原核表达分析。结果表明:1)枸杞查耳酮异构酶LcCHI1基因的cDNA序列全长695 bp,开放阅读框为633 bp,编码210个氨基酸,含有1个保守的Chalcone_3 family结构域。2)氨基酸序列比对显示,LcCHI1蛋白符合Chalcone蛋白结构特征且与其他物种Chalcone蛋白具有高度的相似性。聚类分析表明,LcCHI1蛋白与茄科的CHI蛋白单独聚成一支。3)LcCHI1在枸杞花、嫩叶与嫩枝中表达量较高,在根中表达量较低。随着枸杞果实的发育,LcCHI1表达呈先升高后降低趋势,在变色果中表达量最高。4)在原核表达载体中构建LcCHI1重组蛋白,经SDS-PAGE电泳分析在63 ku处出现表达蛋白。综上,LcCHI1基因在枸杞花和变色果中表达较高,并能进行原核蛋白表达,研究结果为枸杞类黄酮合成途径中CHI基因的功能验证奠定基础。
关键词:  枸杞  查耳酮异构酶  基因克隆  基因表达  原核表达
DOI:10.11841/j.issn.1007-4333.2024.04.17
投稿时间:2023-07-12
基金项目:青海省共和县黄河龙羊峡库区林草生态修复综合治理中央财政支持国土绿化示范试点项目(科技支撑)(E6301000076033516);中藏药材绿色高值化与标准化生产技术集成与示范推广项目(2023-NK-P38)
Cloning and expression analysis of Lycium chinense chalcone isomerase LcCHI1 gene
QIAO Feng
(Key Lab of Medicinal Animal and Plant Resources of Qinghai-Tibetan Plateau in Qinghai Province/Institute of Plateau Science and Sustainable Development, Qinghai Normal University, Xining 810008, China)
Abstract:
The expression patterns and functions of chalcone isomerase CHI1 gene from different tissues in Lycium chinense were investigated in this study. LcCHI1 gene (Genbank No. OR026273) was obtained using homologous cloning method. Its physicochemical properties, structure, and evolutionary relationships were analyzed using bioinformatics methods.The tissue expression specificity was analyzed using fluorescence quantitative PCR(qRT-PCR) technology and prokaryotic expression analysis was performed. The results showed that: 1) The LcCHI1 gene had a total length of 695 bp, an open reading frame of 633 bp, encoding 210 amino acids and containing a conserved chalcone_3 family domains. 2) Multiple sequence alignment showed that the LcCHI1 protein conformed to the structural characteristics of chalcone protein and had high similarity with other chalcone proteins. Cluster analysis showed that the LcCHI1 protein and the CHI protein of the Solanaceae family aggregated into a single branch. 3) LcCHI1 was highly expressed in the flowers, tender leaves, and tender branches of L. chinense, but low in the roots. With the fruit development of L. chinense, the expression of LcCHI1 showed a trend of first increasing and then decreasing, with the highest expression in color changing fruits. 4) The LcCHI1 recombinant protein in the prokaryotic expression vector was constructed, and the expressed protein appeared at 63 ku through SDS-PAGE electrophoresis analysis. In summary, the LcCHI1 gene was highly expressed in flowers and color changing fruits from L. chinense, and can undergo prokaryotic protein expression. The results layed the foundation for the functional verification of the CHI gene in the flavonoid synthesis pathway of L. chinense.
Key words:  Lycium chinense  chalcone isomerase  gene cloning  gene expression  prokaryotic expression