Cloning and Expression Analysis of ANS Gene in Cichorium intybus

doi:10.7668/hbnxb.20195328

Abstract

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

CLC Number:

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.

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Figures/Tables 6

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.

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.

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

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).

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.

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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