Identification and expression analysis of TPS family gene in Cannabis sativa L

Abstract

Terpene synthases (TPSs) regulate plant growth, development, and stress response. TPS genes have been identified in Arabidopsis thaliana and Zea mays. Cannabis sativa TPS genes were identified and analyzed using bioinformatics. Genomic data were downloaded from Plant Transcription Factor Database and National Center for Biotechnology Information database, and TPS genes were predicted, analyzed, and visualized using ExPASy, PlantCare, and other online websites along with TBtools, MEGA software, and other software. To verify its role, quantitative real-time polymerase chain reaction (qRT-PCR) tests were conducted. The Cannabis sativa TPS family comprises 41 elements distributed over 8 chromosomes and a single scaffold segment. The isoelectric point varied between 4.96 and 7.03, while the molecular weight spanned from 20705.90 to 102324.64 Da. The majority of genes were found in the cytoplasm and chloroplasts, with the remainder situated in the peroxisome, nucleus, plasma membrane, and mitochondria. Several cis-acting components associated with stress response were present in the gene's upstream promoter region. Data from RNA sequencing and qRT-PCR revealed specific expression of TPS genes in all five organs of female Cannabis sativa plants. Collinearity analysis showed 4 homologous gene pairs between the Cannabis sativa and Arabidopsis thaliana, with many pairs of homologous genes in other species, which was consistent with the dicotyledons evolutionary relationship. Furthermore, some genes may participate in Cannabis sativa growth and development and play a role in secondary metabolite synthesis. Therefore, bioinformatics analysis of the Cannabis sativa TPS gene family provides a theoretical basis for future research on the volatile terpene compounds of Cannabis sativa.

Keywords: Cannabis sativa L; Evolutionary analys; Expression pattern; TPS enzyme gene; qRT-PCR.

Fig. 1

Chromosome distribution and gene replication events of CsTPS gene. A: Details on the positioning of the CsTPS gene on the chromosomes of Cannabis sativa. B: The replication events of TPS genes in Cannabis sativa. The grey lines represent all collinearity genes in the genome, and the black line represents the tandem replication line relationship between CsTPS genes. Gene density data is depicted by the inner pair of circles, with red indicating high gene density and blue denoting low gene density. Gene pairs duplicated in tandem are denoted in black.

Fig. 2

Tertiary structure analysis of TPS proteins.

Fig. 3

TPS from Cannabaceae, including Cannabis sativa and hops, in the subfamilies are more closely related to each other than they are to TPS from other Cannabis sativa angiosperms and are shown as red pentagrams. The colors indicate the subfamily: blue for TPS-a, purple for TPS-b, grey for TPS-c, green for TPS-e/f, and orange for TPS-d.

Fig. 4

TPS genes structures, CDS: sequence of coding, UTR: untranslated region, lines indicate introns.

Fig. 5

CsTPS gene structure and cis element analysis. The phylogenetic tree of TPS proteins, distribution of conserved genes, and the structure and conserved domains of TPS. Introns are shown as black lines.

Fig. 6

Prediction of cis-elements in Cannabis sativa CsTPS gene.

Fig. 7

Collinearity analysis of TPS in Cannabis sativa and two other species, Arabidopsis thaliana, Malus pumila Mill showing the isotropy among them. The blue line represents the collinear region between Cannabis sativa and other chromosomes.

Fig. 8

Protein–protein interaction network of TPS gene family in Cannabis sativa.

Fig. 9

Selective splicing isomers of 41 CsTPS genes and heat map of gene expression of different isomers in different tissues.

Fig. 10

qRT-PCR verification of highly expressed CsTPS genes in different issues of Cannabis sativa, TPS17, TPS18, TPS20, TPS21, TPS22, TPS26, and TPS27, with flowers as control; TPS40, TPS41, TPS12, TPS13, and TPS16, with leaves as control. Horizontal coordinates represent different tissue parts, and vertical coordinates represent relative expression levels. Statistical significance was determined using t-test: *p < 0.05 to ****p < 0.0001.