Pyrosequencing of the Camptotheca acuminata transcriptome reveals putative genes involved in camptothecin biosynthesis and transport

Background: Camptotheca acuminata is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. C. acuminata produces the terpenoid indole alkaloid, camptothecin (CPT), which exhibits clinical effects in various cancer treatments. Despite its importance, little is known about the transcriptome of C. acuminata and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database.Results: From a constructed cDNA library of young C. acuminata leaves, a total of 30,358 unigenes, with an average length of 403 bp, were obtained after assembly of 74,858 high quality reads using GS De Novo assembler software. Through functional annotation, a total of 21,213 unigenes were annotated at least once against the NCBI nucleotide (Nt), non-redundant protein (Nr), Uniprot/SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Arabidopsis thaliana proteome (TAIR) databases. Further analysis identified 521 ESTs representing 20 enzyme genes that are involved in the backbone of the CPT biosynthetic pathway in the library. Three putative genes in the upstream pathway, including genes for geraniol-10-hydroxylase (CaPG10H), secologanin synthase (CaPSCS), and strictosidine synthase (CaPSTR) were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in C. acuminata. With respect to the branch pathway of CPT synthesis, six cytochrome P450s transcripts were selected as candidate transcripts by detection of transcript expression in different tissues using qRT-PCR. In addition, one glucosidase gene was identified that might participate in CPT biosynthesis. For CPT transport, three of 21 transcripts for multidrug resistance protein (MDR) transporters were also screened from the dataset by their annotation result and gene expression analysis.Conclusion: This study produced a large amount of transcriptome data from C. acuminata by 454 pyrosequencing. According to EST annotation, catalytic features prediction, and expression analysis, novel putative transcripts involved in CPT biosynthesis and transport were discovered in C. acuminata. This study will facilitate further identification of key enzymes and transporter genes in C. acuminata

A novel multiplex PCR-RFLP method for simultaneous detection of the <it>MTHFR 677 C > T</it>, <it>eNOS +894 G > T</it> and <it>- eNOS -786 T > C</it> variants among Malaysian Malays

<p>Abstract</p> <p>Background</p> <p>Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of <it>eNOS</it> +894 G > T and <it>eNOS −</it>786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and <it>eNOS</it> +894 G > T and <it>eNOS −</it>786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and <it>eNOS</it> +894 G > T and <it>eNOS −</it>786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and <it>eNOS</it> +894 G > T and <it>eNOS −</it>786 T > C were calculated using the Hardy Weinberg equation.</p> <p>Methods</p> <p>The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.</p> <p>Results</p> <p>The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for <it>eNOS</it> +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for <it>eNOS −</it>786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).</p> <p>Conclusions</p> <p>Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the <it>MTHFR 677 C > T</it> and <it>eNOS +894 G > T</it> and <it>eNOS −786 T > C</it> variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the <it>MTHFR 677 C > T</it> and <it>eNOS +894 G > T</it> and <it>eNOS −786 T > C</it> variants.</p