Cloning and expression analysis of Cinnamoyl-CoA reductase (CmCCR) genes in ‘Sanhongmiyou’ (Citrus maxima)

Objective CmCCR gene plays key role in lignin biosynthesis. The role and expression pattern of CmCCR during juice sacs granulation were investigated.

Method In this study, bioinformatics, gene cloning, subcellular localization, and fluorescence quantitative PCR techniques were used to screen and clone the CmCCR gene from ‘Sanhongmiyou’ pomelo, and its expression in juice sacs of fruits at different developmental stages of two varieties (‘Sanhongmiyou’ and ‘Liuyuezaoyihao’ pomelo) were examined. The lignin content in juice sacs was determined using the acetyl bromide method.

Results From the RNA-Seq data of ‘Sanhongmiyou’ juice sacs, 5 CmCCR genes were screened. Based on the correlation between lignin content in mature juice sacs and the transcription levels of CmCCR genes, it was preliminarily determined that CmCCR1 might be involved in juice sacs lignin metabolism. The ORF length of the CmCCR1 gene was 1017 bp. Bioinformatics analysis revealed that the CmCCR1 protein has a molecular weight of 37.5 kDa, an isoelectric point (pI) of 7.06, and belongs to stable hydrophilic proteins. It contains an NAD(P)H/NAD(P)(+) binding (NADB) domain and shares three identical motifs with sweet orange CcCCR1, with similar distribution patterns. Phylogenetic analysis showed that CmCCR1 is most closely related to sweet orange CcCCR1 and both belong to the group of dicotyledonous plants. Subcellular localization results indicated that the CmCCR1 gene is located on the endoplasmic reticulum, consistent with previous software predictions. Real-time quantitative PCR results showed a significant increase in the expression level of the CmCCR1 gene in mature juice vesicles of ‘Sanhongmiyou’, while the relative expression level in juice sacs of ‘Liuyuezaoyihao’ pomelo, which had not accumulated lignin, remained consistently low during development and was significantly lower than that of ‘Sanhongmiyou’ at the same developmental stage. Using transient transformation to introduce CmCCR1 into ‘liuyuezao1hao’ pomelo juice sacs, the results showed that the expression level of the CmCCR1 gene in the transformed juice sacs significantly increased, with the phloroglucinol staining showing a pink color, indicating that there was an accumulation of lignin in the juice cells.

Conclusion The CmCCR1 gene may be involved in regulating the biosynthesis of secondary metabolites such as lignin in the juice sacs of ‘Sanhongmiyou’, and its increased expression level is associated with the occurrence of juice sac granulation.