homologous to CHSs from other plants and subgrouped with Ruta Palmatum CHS protein.
Regarding numbers of intron in CHS gene, Ma et al. (2009) found a novel type III PKS2 gene from Polygonum cuspidatum Sieb. et Zucc with three introns in the ORF sequence. All type III PKS genes in flowering plants including B. rotunda CHS gene had one conserved intron with an exception from Antirrhinum majus CHS gene.
Lei et al. (2010) found a CHS gene from hairy root cultures of Scutellaria viscidula Bunge with 1649bp length including an ORF of 1170bp ORF that corresponded to a deduced protein of 390 amino acids. The molecular mass and pI of the Scutellaria viscidula Bunge CHS were calculated at 42.56kDa and 5.79, respectively, which is close to 43.22kDa and 5.86-7.57 of B. rotunda CHS protein. Multiple sequence alignments showed that Scutellaria viscidula CHS protein had high homology with Scutellaria baicalensis CHS protein.
Zhou et al. (2011) showed that Paeonia suffruticosa cv. Yu Ji Yan Zhuang CHS gene sequence was 1475bp with an ORF of 1185bp encoding for 394 amino acids. The bioinformatic analysis showed that all the conserved active sites and the family signature of CHS protein are present in Paeonia suffruticosa CHS gene, designated as CHS1 gene. Sequence alignment and phylogenetic analysis revealed that the Paeonia suffruticosa CHS1 protein shared high homology with CHS protein from Salicaceae, Malvaceae and Rosaceae plant families and had a typical CHS structure.
Similarly to B. rotunda, Lo et al. (2002) isolated seven CHS genes from Sorghum bicolor. The CHS1-7 were highly conserved and closely related to the C2 and Whp genes from maize. The CHS1-7 genes of Sorghum bicolor were the result of recent gene duplication. Expression studies showed that Sorghum bicolor CHS1-7 genes were not differentially expressed in the various growth and developmental conditions, but Sorghum bicolor CHS8 gene was significantly expressed in the pathogen condition. The Sorghum bicolor CHS8 even formed a distinct subgroup from Sorghum bicolor CHS1-7 genes in the phylogenetic analysis. The substitutions on the active site region distinguished Sorghum bicolor CHS8 from naringenin CHS gene, which means it was evolved in defense related mechanism with new enzymatic function in flavonoids biosynthesis pathway.
Jiang et al. (2006) showed another example of sequence variability of CHS gene from gametophores of Physcomitrella patens, the moss. Genome database and sequence analysis of Physcomitrella patens CHS EST showed a multigene family in the moss.
Out of nineteen putative CHS genes, ten genes were expressed and showed corresponding ESTs in the database, indicating a functional divergence of the multigene CHS genes in the moss. The Physcomitrella patens CHS gene also showed variability at the 3' end, similar to 3' end of B. rotunda CHS variants. Despite the variability, Physcomitrella patens CHS protein utilized p-coumaroyl-CoA as the most preferred substrate, therefore it was suggested that Physcomitrella patens CHS is a naringenin chalcone CHS enzyme. In addition, phylogenetic studies showed Physcomitrella patens CHS gene close to microorganism’s CHS-like genes. Koduri et al. (2010) reported at least seventeen putative CHS superfamily genes exist in the genome of Physcomitrella patens, where each of them particularly showed distinct structure organization. Three of these genes (CHS2b, CHS3, and CHS5) of Physcomitrella patens were found in
plant anther-specific CHS-like enzymes. The Physcomitrella patens CHS superfamily appeared to undergo tandem and segmental duplication events that occurred along with intron gain and loss process and resulted to the superfamily genes structure. Ten CHS genes of the superfamily genes were intron-less. It seems that Physcomitrella patens CHS gene was duplicated at least twice via retrotransposition in the lineage, nevertheless large numbers of Physcomitrella patens CHSs protein had similarity in sequences, catalytic motifs, and EST data encoding for an active CHS enzyme. The Physcomitrella patens CHS superfamily genes showed differential regulation with respond to light.
The genome of Oryza sativa had been completely sequenced and helped to investigate genes containing typical CHS domains. Han et al. (2009) found a CHS multigene family in Oryza sativa rice genome. Sequence and functional divergence of CHS gene repeatedly occurred in many plants resulted CHS multigene family genes. The Oryza sativa CHS superfamily members were diverged into four branches, which every of particular branch have specific function. Two conserved CHS genes were clustered with CHS genes from monocotyledon and dicotyledon species and considered true CHS genes involved in flavonoids and anthocyanin biosynthesis, however other two CHS genes in a distant branch showed fertility functions. Many uncertain CHS genes were clustered with fatty acid synthase genes. More investigation on B. rotunda genome could help to discover the potential CHS genes and to accurately determine the arrangement and copy number of the CHS gene in the genome.
Transcriptome analysis of B. rotunda CHS gene showed twenty-six unigenes, which are potential to be CHS gene. Fourteen of theses unigenes showed the sequence of CHS gene based on Blast alignment score, while others were not identified as CHS gene.
Among fourteen unigenes, six of them showed the sequence of B. rotunda CHS gene,
which might be due to different expression pattern in B. rotunda tissues as Phe-treated callus was sent for transcriptome analysis while rhizome was used in RACE analysis.
The sequence of two unigenes was completely matched with the sequence of B. rotunda CHS gene indicating the presence of B. rotunda CHS variants in transcriptome. Variants of 1-4 of B. rotunda CHS gene were found among the unigenes indicating that these variants were expressed in the treated callus.
Through transcriptome analysis Zabala et al. (2006) studied genes involved in the phenylpropanoid pathway in soybean leaves in response to infection with Pseudomonas syringae pv. Glycinea. Although CHS transcripts highly accumulate after inoculation of soybean plants but they found that all CHS transcripts except CHS4 member in the eight-member gene family were highly accumulated during defense response in soybean. The CHS gene was determined as an early responsive gene in defense mechanism in soybean. Transcriptome screening seems to verify the variants of B.
rotunda CHS gene among the unigenes, however further analysis is required to explore the variability of B. rotunda CHS genes.