菊苣<i>ANS</i>基因的克隆及表达分析

doi:10.7668/hbnxb.20195328

摘要/Abstract

摘要：

花青素合酶(ANS)是植物花青素合成过程中的关键酶,可以将无色的花色苷催化成红色、橙色、蓝色等不同颜色的花青素。为了探究ANS基因调控菊苣叶色的分子机制,首先对该基因进行生物信息学分析,随后选取红色结球型菊苣(印地欧)和绿色菊苣(普那)为材料,克隆得到菊苣ANS基因(CiANS),并对该基因在这2个材料中的氨基酸序列及蛋白结构进行了差异分析,最后对ANS基因的组织特异表达和蛋白的亚细胞定位进行了检测,并对ANS蛋白进行了原核表达。结果显示,CiANS与莴苣及栽培菊苣的ANS亲缘关系最近,Motif分析发现,CiANS蛋白基序在不同植物中均较为保守。2种菊苣中ANS的克隆结果表明,ANS基因全长均为1 068 bp,编码355个氨基酸,具有9个差异氨基酸,但在预测的三级结构上并未表现出明显的差异。qRT-PCR结果显示,CiANS基因在不同组织中均有表达,但在叶片中表达量最高,并且红色的结球菊苣中CiANS基因的表达水平显著高于绿色的普那菊苣。亚细胞定位结果显示,CiANS蛋白定位于细胞质和细胞核中。CiANS蛋白在原核载体上诱导表达后大小共为45 ku,与预期蛋白大小一致。综上所述,菊苣叶色差异的形成很可能与ANS基因的表达量差异有关,研究结果为解析菊苣叶色分子调控网络及花青素代谢遗传改良提供了理论依据。

关键词: 菊苣, 花青素合酶, 表达分析, 亚细胞定位, 原核表达

Abstract:

Anthocyanin synthase(ANS)is a key enzyme in the anthocyanin biosynthesis pathway in plants,catalyzing the conversion of colorless leucoanthocyanidins into colorful anthocyanins such as red,orange,and blue.To investigate the molecular mechanism of ANS gene in leaf coloration regulation in chicory(Cichorium intybus),bioinformatics analysis of ANS was first conducted.Then,the red heading C.intybus(Indiou)and green forage variety(Puna)were selected as the materials to clone the C.intybus ANS (CiANS)gene.Differences in amino acid sequences and protein structures of CiANS between the two materials were analyzed.Finally,tissue-specific expression of the ANS gene and subcellular localization of CiANS were characterized,and prokaryotic expression of the CiANS was performed.The results showed that CiANS shared the closest phylogenetic relationship with ANS from lettuce(Lactuca sativa)and endive(C.endivia).Motif analysis revealed that the protein motifs of CiANS were relatively conserved across different plant species.Cloning results indicated that the full-length ANS gene in both chicory varieties was 1 068 bp,encoding 355 amino acids with 9 divergent residues,though no significant differences were observed in predicted tertiary structures.qRT-PCR results demonstrated that CiANS was expressed in all tissues,with the highest expression level in leaves,and its expression in red heading Indiou was significantly higher than in green Puna.Subcellular localization revealed that CiANS protein was localized in both the cytoplasm and nucleus.After prokaryotic expression,the induced CiANS protein exhibited a molecular weight of 45 ku,consistent with the predicted size.In conclusion,the observed leaf color variation in C.intybus is likely associated with differential expression levels of the CiANS gene.This study provides theoretical insights for elucidating the molecular regulatory network of Cichorium intybus leaf coloration and genetic improvement of anthocyanin metabolism.

Key words: Cichorium intybus, Anthocyanin synthase, Expression analysis, Subcellular localization, Prokaryotic expression

中图分类号:

冯清香, 朱哈修, 杜洋, 徐艳茹, 王晨晨, 刘雪, 李大勇, 周军, 张彬. 菊苣ANS基因的克隆及表达分析[J]. 华北农学报, 2025, 40(3): 51-59. doi: 10.7668/hbnxb.20195328.

FENG Qingxiang, ZHU Haxiu, DU Yang, XU Yanru, WANG Chenchen, LIU Xue, LI Dayong, ZHOU Jun, ZHANG Bin. Cloning and Expression Analysis of ANS Gene in Cichorium intybus[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 51-59. doi: 10.7668/hbnxb.20195328.

http://www.hbnxb.net/CN/Y2025/V40/I3/51

图/表 6

图1 CiANS与其他物种ANS蛋白系统进化树及motif预测 A.菊苣及其他物种的ANS系统进化树;B.菊苣及其他物种ANS motif预测。

Fig.1 Phylogenetic tree and motif prediction of ANS protein in Cichorium intybus and other species A.ANS phylogenetic tree of Cichorium intybus and other species;B.Prediction of the ANS motif in Cichorium intybus and other species.

图2 CiANS编码区PCR扩增及氨基酸序列比对 A.CiANS基因PCR扩增条带:M.DNA 2K Marker;B.CiANS氨基酸序列比对。

Fig.2 PCR amplification of the CiANS coding region and subsequent amino acid sequence alignment A.PCR-amplified band of the CiANS gene:M.DNA 2K Marker;B.Comparison of the CiANS amino acid sequence.

图3 CiANS蛋白三级结构预测 A.普那菊苣;B.印地欧菊苣。

Fig.3 Prediction of the tertiary structure of CiANS protein A.Puna Cichorium intybus;B.Indiou Cichorium intybus.

图4 CiANS基因在菊苣不同组织中的表达量 A.2种菊苣的叶片颜色表型;B.2种菊苣不同组织中CiANS的表达量分析。不同小写字母代表差异显著(P<0.05)。

Fig.4 Expression of CiANS gene in different tissues of Cichorium intybus A.Leaf color phenotypes of 2 varieties of Cichorium intybus;B.Expression of CiANS in different tissues of 2 varieties of Cichorium intybus.Different lowercase letters represent significant different (P<0.05).

图5 CiANS蛋白亚细胞定位 Bright.明场照片;GFP.载体上绿色荧光蛋白在莴苣原生质体中定位的照片;RFP.载体上红色荧光蛋白在莴苣原生质体中定位的照片;Merge.GFP及RFP通道叠加的照片。

Fig.5 Subcellular localization of CiANS protein Bright.Bright field image;GFP.Localization of green fluorescent protein on the carrier in Lactuca sativa protoplasts;RFP.Localization of red fluorescent protein on the carrier in Lactuca sativa protoplasts;Merge.Composite image combining the GFP and RFP channels.

图6 CiANS原核表达和纯化 M.MF212 蛋白Marker(10~180 ku);1,2.空载体表达后纯化前的上清和沉淀;3,4.pET-28a-CiANS诱导前的上清和沉淀;5,6.pET-28a-CiANS诱导后纯化前的上清和沉淀;7,8.pET-28a-CiANS诱导后纯化过程中的洗脱液;9.pET-28a-CiANS纯化后的蛋白。

Fig.6 Prokaryotic expression and purification of CiANS M.MF212 protein Marker(10—180 ku);1,2.Supernatant(labeled 1)and precipitation(labeled 2)before purification after expression of pET-28a empty vector;3,4.Supernatant(labeled 3)and precipitation(labeled 4)before induction of pET-28a-CiANS;5,6.Supernatant(labeled 5)and precipitation(labeled 6)before purification after induction of pET-28a-CiANS;7,8.Eluate during purification after induction of pET-28a-CiANS;9.Protein after pET-28a-CiANS purification.

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菊苣ANS基因的克隆及表达分析

Cloning and Expression Analysis of ANS Gene in Cichorium intybus

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