Welcome to Chinese Journal of Tropical Crops,

Chinese Journal of Tropical Crops ›› 2022, Vol. 43 ›› Issue (12): 2422-2430.DOI: 10.3969/j.issn.1000-2561.2022.12.004

• Omics & Biotechnology • Previous Articles     Next Articles

Cloning and Bioinformatics Analysis of Pepper PnCAD Gene by Race

SUN Yeqiao1,2,3, WANG Jue1,2,3, HU Lisong2,3, WU Baoduo2,4, HAO Chaoyun2,4,5, FAN Rui2,4,5,*()   

  1. 1. College of Tropical Crops, Hainan University, Haikou, Hainan 570228, China
    2. Institute of Spices and Beverages, Chinese Academy of Tropical Agricultural Sciences, Wanning, Hainan 571533, China
    3. Hainan Key Laboratory of Genetics, Breeding and Germplasm Resources, Wanning, Hainan 571533, China
    4. Hainan Key Laboratory of Genetic Improvement and Quality Control of Tropical Spice Beverage Crops, Wanning, Hainan 571533, China
    5. Hainan SIM Soonliang Academician Workstation, Wanning, Hainan 571533, China
  • Received:2022-03-28 Revised:2022-06-23 Online:2022-12-25 Published:2023-01-12
  • Contact: FAN Rui


Lignin has some functions in plants, such as transporting water, supporting plants and strengthening plants against damage, etc. It is one of the important products of phenylpropane metabolism. Among them, cinnamyl alcohol dehydrogenase (CAD) is an important rate-limiting enzyme in the lignin synthesis pathway. In this experiment, on the basis of pepper transcriptome sequencing, the RACE method was used for gene cloning, bioinformatics analysis of the full-length PnCAD gene, and many analyses of its protein such as physicochemical properties, subcellular localization and phylogenetic tree. It was analyzed by qPCR. The cinnamyl alcohol dehydrogenase gene was cloned by the race method. Finally, a full-length cDNA with a length of 1364 bp was obtained, including 1071 bp open reading frame (ORF), encoding 356 amino acids. The predicted relative molecular weight was 3.879 kDa and the isoelectric point was 6.27. It belonged to hydrophilic protein. It contained three N-glycosylation characteristic sequences and nine phosphorylation sites, which were likely to be in the cytoplasm. Domain analysis showed that CAD protein contained NAD (P) binding sites, multiple catalytic zinc and structural zinc binding sites. Phylogenetic tree analysis showed that pepper CAD was closely related to Asarum CAD 6, and the highest homology was 76%, both of which belonged to primitive dicotyledons. Through fluorescence quantitative analysis, it could be found that the expression of P. flaviflorum under the infection of Phytophthora capsici increased, reached the highest value at 8 h, about 10 times that of the control, then increased slightly at 24 h, about 6 times that of the control group, and then decreased. Generally speaking, the gene expression level of P. flaviflorum at all time was higher than that of P. nigrum cv. Reyin-1 and was significantly different. The experimental data could provide reference data for the future study of the anti abiotic stress function of black pepper, and also provide a theoretical basis for the functional study of gene PnCAD.

Key words: Piper nigrum, PnCAD, gene cloning, bioinformatics analysis, qPCR

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