Cloning，Structure and Expression Analysis of Chalcone Synthase Gene from Carthamus tinctorius L.

doi:10.15933/j.cnki.1004-3268.2022.09.006

Abstract

Abstract: Chalcone synthase（CHS）is the key rate⁃limiting enzyme in the biosynthesis of flavonoids，which are the main bioactive components of Carthamus tinctorius L..In order to elucidate the synthetic pathway of flavonoids in Carthamus tinctorius L.，the cDNA sequence and gDNA sequence of CtCHS gene were obtained by RT⁃PCR and PCR using safflower cultivar as material，the gene structure，protein structure，phylogeny were analyzed，and the expression patterns of CtCHS in different tissues，and underdifferent exogenous hormone treatments were detected by qRT⁃PCR. The results showed that the length of CtCHS cDNA was 1 317 bp，and gDNA was 1 815 bp，which consisted of two exons and one intron.The content of AT in intron was significantly higher than that of GC，and was significantly higher than that of AT in exon.In addition，the intron sequence contained cis acting elements similar to enhancers，light and drought stress responsers，which might play a role in enhancing gene transcription and might be induced by light or drought stress.The cDNA sequence of CtCHS gene contained a maximum open reading frame of 1 206 bp，encoding 401 amino acids.The corresponding protein was hydrophilic protein，had no signal peptide sequence and transmembrane structure，and was localized in cytoplasm. The results of phylogenetic analysis of CHS in pharmaceutical plants showed that the CtCHS protein had the closest evolutionary relationship with CHS of Centaurea cyanus，but had a distant evolutionary relationship with CHS of Bletilla striata and Dendrobium candidum.Besides CHS proteins of pharmaceutical plants had obvious species characteristics.The results of qRT⁃PCR showed that the expression of CtCHS was the highest in flowers and the lowest in early fruit balls and young roots.At different stages of flower development，the expression level of CtCHS gene showed the same trend in the two Carthamus tinctorius L.varieties（red and white cultivars），and both had the highest expression level at blooming stage.However，the red cultivars had the lowest expression level at late stage of flower tissue formation，while the white cultivar had the lowest expression level at petal initial extending stage.In addition，the expression of CtCHS gene was significantly different between red and white safflower cultivars in initial stage of flower tissue formation，late stage of flower tissue formation，young stems and young leaves，and the difference in petal basically extending stage was extremely significant，while the difference in other stages and other tissues was not significant.The expression analysis of CtCHS after hormone stress showed that CtCHS could respond to different exogenous hormone treatments，thus affecting its expression.

Key words: Carthamus tinctorius L. , Chalcone synthase, Gene structure, Intron, Expression analysis

摘要： 黄酮类物质是红花的主要活性成分，查尔酮合成酶（Chalcone synthase，CHS）是黄酮类物质合成过程中的关键限速酶。为解析红花黄酮类物质的合成途径，以大果球红花为材料，通过逆转录PCR（RT-PCR）及PCR技术克隆红花CHS（CtCHS）基因的cDNA和gDNA序列，分析其基因结构、蛋白质结构和系统进化关系，采用实时荧光定量PCR（qRT-PCR）方法分析CtCHS 基因的组织表达模式及激素处理下的表达模式。结果表明，CtCHS基因的cDNA序列长1 317 bp，gDNA序列长1 815 bp，由2个外显子和1个内含子构成。内含子AT含量明显高于GC含量，且明显高于外显子中AT含量，内含子序列含有类似增强子及光照、干旱胁迫响应的顺式作用元件，可能在增强基因转录中发挥作用，并可能受光照、干旱等胁迫的诱导。该基因的cDNA序列包含1个1 206 bp的最大开放阅读框，编码401个氨基酸，相应的蛋白质为亲水性蛋白，无信号肽序列，不存在跨膜结构，定位于细胞质。药用植物CHS系统进化分析结果表明，CtCHS与矢车菊CHS的进化关系最近，与白芨、铁皮石斛等CHS的进化关系较远，且药用植物CHS蛋白具有明显的种属特性。qRT-PCR检测结果表明，CtCHS在花中的表达量最高，初期果球和幼根中的表达量最低。不同花期，红色和白色红花中CtCHS基因的表达量呈现相同的表达趋势，均在盛花期表达量最高，但红色红花在花组织形成后期表达量最低，而白色红花在花瓣开始伸出期表达量最低。另外，CtCHS基因的表达量在红色和白色红花花组织形成初期、后期及幼茎和幼叶中差异显著，在花瓣基本伸出期差异极显著，而在其他时期、其他组织中差异不显著。激素胁迫后的表达量检测结果表明，CtCHS可响应不同外源激素处理，从而影响其表达。

关键词: 红花, 查尔酮合成酶, 基因结构, 内含子, 表达分析

CLC Number:

S567.2

LU Dandan, TAN Zhengwei, YANG Hongqi, LI Lei, YU Yongliang, XU Lanjie, YANG Qing, LIANG Huizhen. Cloning，Structure and Expression Analysis of Chalcone Synthase Gene from Carthamus tinctorius L.[J]. Journal of Henan Agricultural Sciences, 2022, 51(9): 56-67.

鲁丹丹, 谭政委, 杨红旗, 李磊, 余永亮, 许兰杰, 杨青, 梁慧珍. 红花查尔酮合成酶基因的克隆、结构及表达分析[J]. 河南农业科学, 2022, 51(9): 56-67.

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