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Computational Identification and Systematic Classification of Novel Cytochrome P450 Genes in Salvia miltiorrhiza.

Chen H, Wu B, Nelson DR, Wu K, Liu C - PLoS ONE (2014)

Bottom Line: The RNA-Seq results showed that 35 CYP450 genes were co-expressed with CYP76AH1, a marker gene for tanshinone biosynthesis, using r≥0.9 as a cutoff.Comparing against the KEGG database, 10 CYP450 genes were found to be associated with diterpenoid biosynthesis.Moreover, we found that 15 CYP450 genes were possibly regulated by antisense transcripts (r≥0.9 or r≤-0.9).

View Article: PubMed Central - PubMed

Affiliation: Center for Bioinformatics, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
Salvia miltiorrhiza is one of the most economically important medicinal plants. Cytochrome P450 (CYP450) genes have been implicated in the biosynthesis of its active components. However, only a dozen full-length CYP450 genes have been described, and there is no systematic classification of CYP450 genes in S. miltiorrhiza. We obtained 77,549 unigenes from three tissue types of S. miltiorrhiza using RNA-Seq technology. Combining our data with previously identified CYP450 sequences and scanning with the CYP450 model from Pfam resulted in the identification of 116 full-length and 135 partial-length CYP450 genes. The 116 genes were classified into 9 clans and 38 families using standard criteria. The RNA-Seq results showed that 35 CYP450 genes were co-expressed with CYP76AH1, a marker gene for tanshinone biosynthesis, using r≥0.9 as a cutoff. The expression profiles for 16 of 19 randomly selected CYP450 obtained from RNA-Seq were validated by qRT-PCR. Comparing against the KEGG database, 10 CYP450 genes were found to be associated with diterpenoid biosynthesis. Considering all the evidence, 3 CYP450 genes were identified to be potentially involved in terpenoid biosynthesis. Moreover, we found that 15 CYP450 genes were possibly regulated by antisense transcripts (r≥0.9 or r≤-0.9). Lastly, a web resource (SMCYP450, http://www.herbalgenomics.org/samicyp450) was set up, which allows users to browse, search, retrieve and compare CYP450 genes and can serve as a centralized resource.

No MeSH data available.


Validation of the expression patterns of 18 randomly selected CYP450 genes and CYP76AH1 across the three tissues of S. miltiorrhiza.Fold changes of transcript levels in flower, leaf and root tissues of S. miltiorrhiza are shown. The average expression levels in three tissues were arbitrarily set to 1. The error bars represent the standard error relative to the mean (SEM). ‘#’ indicates that the genes did not show similar relative expression levels between RNA-Seq results and qRT-PCR results.
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pone-0115149-g007: Validation of the expression patterns of 18 randomly selected CYP450 genes and CYP76AH1 across the three tissues of S. miltiorrhiza.Fold changes of transcript levels in flower, leaf and root tissues of S. miltiorrhiza are shown. The average expression levels in three tissues were arbitrarily set to 1. The error bars represent the standard error relative to the mean (SEM). ‘#’ indicates that the genes did not show similar relative expression levels between RNA-Seq results and qRT-PCR results.

Mentions: The expression levels (FPKM) of the 116 full-length CYP450 genes were calculated using the Trinity program [30] and are shown in S4 Table. To validate the expression levels of CYP450 genes obtained from the RNA-Seq experiment, we performed qRT-PCR on 18 randomly selected CYP450 genes and SmCYP76AH1. RNAs were extracted from three biological replicates for each tissue type, and each biological replicate had three technical replicates for the qRT-PCR experiments. For each gene, the mean expression level of all biological replicates and technical replicates for each tissue type was calculated. Two methods were used to compare the RNA-Seq and qPCR results. First, the mean expression levels in the three tissue types for each gene were ordered. If the order of expression levels obtained from the RNA-Seq result was the same as that from the qRT-PCR result, the RNA-Seq result was considered to be validated, As shown in Fig. 7, 15 of 18 (78.9%) results were validated. Second, for each gene, the average expression levels for the three biological replicates were calculated. Then, the Pearson correlation coefficients of the tissue expression profiles of each gene obtained by RNA-Seq and qPCR were calculated. The results are shown in S5 Table, and eleven gene pairs had r> = 0.9. These data suggested that the expression patterns deduced from the FPKM values in our transcriptome analyses were reliable and can be used in downstream gene expression analyses.


Computational Identification and Systematic Classification of Novel Cytochrome P450 Genes in Salvia miltiorrhiza.

Chen H, Wu B, Nelson DR, Wu K, Liu C - PLoS ONE (2014)

Validation of the expression patterns of 18 randomly selected CYP450 genes and CYP76AH1 across the three tissues of S. miltiorrhiza.Fold changes of transcript levels in flower, leaf and root tissues of S. miltiorrhiza are shown. The average expression levels in three tissues were arbitrarily set to 1. The error bars represent the standard error relative to the mean (SEM). ‘#’ indicates that the genes did not show similar relative expression levels between RNA-Seq results and qRT-PCR results.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262458&req=5

pone-0115149-g007: Validation of the expression patterns of 18 randomly selected CYP450 genes and CYP76AH1 across the three tissues of S. miltiorrhiza.Fold changes of transcript levels in flower, leaf and root tissues of S. miltiorrhiza are shown. The average expression levels in three tissues were arbitrarily set to 1. The error bars represent the standard error relative to the mean (SEM). ‘#’ indicates that the genes did not show similar relative expression levels between RNA-Seq results and qRT-PCR results.
Mentions: The expression levels (FPKM) of the 116 full-length CYP450 genes were calculated using the Trinity program [30] and are shown in S4 Table. To validate the expression levels of CYP450 genes obtained from the RNA-Seq experiment, we performed qRT-PCR on 18 randomly selected CYP450 genes and SmCYP76AH1. RNAs were extracted from three biological replicates for each tissue type, and each biological replicate had three technical replicates for the qRT-PCR experiments. For each gene, the mean expression level of all biological replicates and technical replicates for each tissue type was calculated. Two methods were used to compare the RNA-Seq and qPCR results. First, the mean expression levels in the three tissue types for each gene were ordered. If the order of expression levels obtained from the RNA-Seq result was the same as that from the qRT-PCR result, the RNA-Seq result was considered to be validated, As shown in Fig. 7, 15 of 18 (78.9%) results were validated. Second, for each gene, the average expression levels for the three biological replicates were calculated. Then, the Pearson correlation coefficients of the tissue expression profiles of each gene obtained by RNA-Seq and qPCR were calculated. The results are shown in S5 Table, and eleven gene pairs had r> = 0.9. These data suggested that the expression patterns deduced from the FPKM values in our transcriptome analyses were reliable and can be used in downstream gene expression analyses.

Bottom Line: The RNA-Seq results showed that 35 CYP450 genes were co-expressed with CYP76AH1, a marker gene for tanshinone biosynthesis, using r≥0.9 as a cutoff.Comparing against the KEGG database, 10 CYP450 genes were found to be associated with diterpenoid biosynthesis.Moreover, we found that 15 CYP450 genes were possibly regulated by antisense transcripts (r≥0.9 or r≤-0.9).

View Article: PubMed Central - PubMed

Affiliation: Center for Bioinformatics, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
Salvia miltiorrhiza is one of the most economically important medicinal plants. Cytochrome P450 (CYP450) genes have been implicated in the biosynthesis of its active components. However, only a dozen full-length CYP450 genes have been described, and there is no systematic classification of CYP450 genes in S. miltiorrhiza. We obtained 77,549 unigenes from three tissue types of S. miltiorrhiza using RNA-Seq technology. Combining our data with previously identified CYP450 sequences and scanning with the CYP450 model from Pfam resulted in the identification of 116 full-length and 135 partial-length CYP450 genes. The 116 genes were classified into 9 clans and 38 families using standard criteria. The RNA-Seq results showed that 35 CYP450 genes were co-expressed with CYP76AH1, a marker gene for tanshinone biosynthesis, using r≥0.9 as a cutoff. The expression profiles for 16 of 19 randomly selected CYP450 obtained from RNA-Seq were validated by qRT-PCR. Comparing against the KEGG database, 10 CYP450 genes were found to be associated with diterpenoid biosynthesis. Considering all the evidence, 3 CYP450 genes were identified to be potentially involved in terpenoid biosynthesis. Moreover, we found that 15 CYP450 genes were possibly regulated by antisense transcripts (r≥0.9 or r≤-0.9). Lastly, a web resource (SMCYP450, http://www.herbalgenomics.org/samicyp450) was set up, which allows users to browse, search, retrieve and compare CYP450 genes and can serve as a centralized resource.

No MeSH data available.