| This study systematically studied the sequence characteristics, chromosomal location, phylogeny, conserved motifs, gene structure and gene expression of HD-Zip transcription factors in the cannabis genome. The real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method was used. (Using StarLighter product: (StarLighter High-Performance Dye-Based qPCR Master Mix - Universal) Verify its functionality. By identifying their domain structures, 33 members of the cannabis HD-ZIP family were identified. Based on their chromosomal localization, these 33 members were named CsHD1-CsHD33 (Figure 1). Within the cannabis HD-ZIP family, each HD-ZIP gene has a matching chromosomal location, evenly distributed across all ten chromosomes.  |
| Figure 1. Chromosomal localization of the CsHD gene. Chromosome length (bp) is represented on the y-axis. Gene concentration is depicted by colored stripes on each chromosome. |
| Analysis of the cannabis HD-Zip gene family members using relevant analysis sites (Table 1) revealed relative molecular masses ranging from 23,264.32 Da to 93,147.87 Da, amino acid counts ranging from 136 to 849 aa, and isoelectric points ranging from 4.54 to 9.04. The results indicate that all cannabis HD-Zip proteins are hydrophilic, but with varying degrees of hydrophilicity. The expected subcellular localization of the cannabis HD-Zip gene family members confirmed that, with the exception of CsHD20, which is localized to the cytoplasm, all proteins are localized to the nucleus. These results may provide a theoretical basis for studying the functional role of the cannabis HD-Zip gene family. |
 |
| Table 1. Information and characteristics of the CsHD gene family |
| The article constructed a neighbor-joining phylogenetic tree using 33 HD-Zips from cannabis and 48 HD-Zips from Arabidopsis (Figure 2). The cannabis HD-Zip family was identified as being divided into four subfamilies: HD-Zip I family (14 genes), HD-Zip II family (7 genes), HD-Zip III family (3 genes), and HD-Zip IV family (9 genes). Covariance analysis among cannabis, rice, and Arabidopsis revealed that the cannabis HD-ZIP family shares 20 orthologous genes with Arabidopsis and rice, 12 of which are shared (Figure 3). Homologous genes in different species maintain identical or similar functions during evolution, and tissue expression patterns of genes are often correlated with gene function. |
 |
| Figure 2. Phylogenetic analysis of the cannabis and Arabidopsis homeodomain-leucine zipper (HD-Zip) families. |
 |
|
Figure 3. Collinearity analysis of homeodomain-leucine zipper (HD-Zip) genes among cannabis, rice, and Arabidopsis. Colored lines indicate co-linked regions between cannabis chromosomes and other chromosomes. |
|
Online analysis using the MEME website and visualization using TBtools software (Figure 4) revealed that proteins in the cannabis HD-Zip gene family contain multiple motifs. Motifs 1 and 2 form the homeodomain and LZ domain, respectively, indicating that motifs 1 and 2 are characteristic of proteins in the cannabis HD-Zip gene family. Members of the same subfamily share similar motif types and numbers. Intron and exon analysis revealed that while members of the same subfamily have relatively consistent intron numbers, there is significant variation in the number of introns and exons between members of different subfamilies. This variation in the number of introns and exons within cannabis HD-Zip genes reflects the structural diversity among members of the cannabis HD-Zip family, while exon numbers are more consistent among members of the same clade in the evolutionary tree. |
|
|
|
Figure 4. Structure of the HD-Zip gene. A. Phylogenetic tree of CsHD proteins. B. Conserved domains of CsHD proteins. C. CsHD gene structure and conserved domains. Introns are indicated by black lines. |
|
The presence of distinct cis-acting elements in the promoter region often indicates that the gene is responsive to hormones or stresses. In the cannabis HD-Zip gene family, each gene promoter region contains cis-elements, with light-responsive elements being the most numerous (Figure 5), followed by hormone-like cis-elements. Analysis of the expression patterns of members of the cannabis HD-Zip gene family in different tissues revealed that 23 genes are highly expressed in flowers (Figure 6). |
|
|
|
Figure 5. Distribution of cis-acting elements in the CsHD-Zip gene. Distribution of 21 cis-acting elements in 33 CsHD-Zip promoter regions. These elements are represented by different symbols. |
|
|
|
Figure 6. CsHD gene expression analysis |
|
The authors sequenced the transcriptomes of five cannabis tissue sites, including flowers, stems, leaves, bracts, and seeds, and found that the expression of 33 cannabis HD-Zip gene family members was convergent among the five tissue sites (Figure 7). |
|
|
|
Figure 7. Differential expression analysis of CsHD family genes in different tissues of cannabis. |
|
In summary, this article systematically investigated the sequence characteristics, chromosomal location, gene structure, conserved sequences, phylogeny, and differential gene expression of the HD-Zip transcription factor in the cannabis genome. These results suggest that HD-Zip genes in cannabis exhibit tissue-specific expression and may be influenced by hormones and external environmental factors. This study contributes to a more comprehensive understanding of the biological functions of the cannabis gene family and lays the foundation for further research on HD-Zip gene function and cannabis cultivar improvement. |
|
Product Ordering Information |
|
|
