Identification and analysis of the R2R3-MYB transcription factor based on a transcriptome database in Morinda officinalis / 药学学报

In order to explore MYB transcription factors related to developmental processes and secondary metabolism in Morinda officinalis, we analyzed MoMYB expression based on transcriptome data from three tissues (root, stem and leaf). We used this analysis to provide a theoretical foundation for regulating the metabolism of M. officinalis. RNA-seq data along with the five databases including PFAM and plantTFDB and others were used to screen and classify MoMYB, including GO functional annotation and classification, subcellular localization, signal peptide prediction, conserved motif discovery, and comparative phylogenetic analysis. RT-qPCR was carried out to detect tissue-specific expression differences of MoMYB genes. According to transcriptome data, 109 MoMYB sequences were identified and divided into four classes, containing 51 sequences related to R2R3-MYB. Subcellular localization analysis indicated that a majority of sequences were located in nucleus. Blast2GO analysis showed that 109 MoMYB sequences were classified into three major functional ontologies including molecular function (112), biological processes (76) and cellular components (239). The R2-MYB conserved motif of 51 R2R3-MYB sequences possessed three significantly conserved tryptophan residues, whereas a phenylalanine replaced the first tryptophan in R3-MYB. The results of multiple sequence alignment and phylogenetic analysis revealed that the R2R3-MYB was distributed in all subgroups, apart from the S10, S19 and S21 subgroups. RT-qPCR indicated that several R2R3-MYB genes were differentially expressed among the three tissues, and this finding was consistent with transcriptome data. The 109 MoMYB sequences were annotated and divided into different classes, which lays the foundation for further study on MYB transcriptional factors in M. officinalis.

Cloning of MYB gene and construction of greenfluorescent protein expression vector in Erigeron breviscapus / 中草药

Objective To study the role of MYB transcription factor in phenylpropane biosynthesis pathway and the response to biotic or abiotic stress, a full length sequence of putative MYB gene was cloned in Erigeron breviscapus. Methods Based on a partial sequence of putative MYB gene from transcriptome we have previous reported, the full length cDNA was cloned by RACE method in E. breviscapus. According to the sequence of cloning cDNA, the nucleotide sequence similarity, physicochemical properties, hydrophobicity, transmembrane structure, secondary structure and tertiary structure were predicted and analyzed by various softwares. Meanwhile, multiple sequence alignment of cloned MYB gene was performed, and the phylogenetic tree was constructed. In addition, the fusion expression vector of this gene and green fluorescent protein was constructed. Results The MYB gene was cloned and named eBMYB06. The open reading frame was 783 bp, encoding 260 amino acid residues with a relative molecular mass of 63.80 kDa and a theoretical pI of 5.18 which proved to be a stable protein. The secondary structure of the protein is mainly composed of irregular coil, alpha helix and beta fold. According to the result of phylogenetic tree alignment of E. breviscapus with R2R3MYBs from Arabidopsis thaliana, the cloned MYB gene was clustered to two subsets of R2R3-MYB genes from A. thaliana, which suggested that the cloned MYB gene would be similar with the two groups in structure and function, which would be involved in response to biotic and abiotic stress or phenylpropane biosynthesis pathway, respectively. Further experimental results show that the constructed expression can be used for the efficient transformation in E. breviscapus. Conclusion For the first time, a MYB gene was cloned may be involved in the phenylpropanoid metabolism or gene metabolism or gene regulation in response to the environment in E. breviscapus.

Molecular characterization of a cDNA encoding caffeoyl-coenzyme A 3 -O- methyltransferase of Stellaria longipes.

A cDNA clone encoding S-adenosyl-L-methionine: trans-caffeoyl-CoA 3-O-methyltransferase (EC 2·1·1·104; CCoAOMT) from Stellana longipes Goldie (long-stalked chickweed) was isolated and studied. Structural analysis of both the nucleotide sequence and the predicted amino acid sequence suggests that our cloned sequence encoded a CCoAOMT enzyme of Stellaria longipes, which shared overall structural similarity with other plant CCoAOMTs but exhibited certain distinct characteristics. Southern blot hybridization and cloning analyses indicating a small CCoAOMT gene family in the Stellana longipes genome and the absence of introns in the coding region of the cDNA- corresponding gene. Sequence variations in the coding region were found among three genotypes from geographically isolated populations. Higher levels of CCoAOMT mRNA were detected in stems and leaves than in roots. The cDNAencoded protein expressed in Eschendia coli was shown to utilize caffeoyl-CoA, but not caffeic acid or 5-hydroxy ferulic acid, as its substrate.