Expression and purification of coat protein of citrus tristeza virus

Citrus tristeza virus (CTV) polyclonal antibodies produced either from the recombinant coat protein (CP) of CTV or extracted virus from midrib used for the detection of virus. Compared with intact virion procedure, the use of CP antigen resulted in highly specific polyclonal antibodies. CTV coat protein gene (CTV-cp) cloned in pQE30 vector and transformed to DH5α containing 666bp long from Thailand MK-50 isolate was amplified with a forward primer CTV-CP1 (5’ CAC CGA CGA AAC AAA GAA ATT GAA GAA CA 3’) and a reverse primer CTVCP2 (5’ TCA ACG TGT GTT AAA TTT CCC AAG C 3’) and cloned into TOPO vector and transformed to TOP10 E. coli competent cell. Six colonies of TOP10 E. coli were selected and checked for the appropriate insertion of cp gene with PCR using T7F (5’ TAA TAC GAC TCA CTA TAG GG 3’) as forward primer and CTVCP2 as reverse primer. Two colonies having appropriate insertion were selected for transformation into BLD21 star (DE3) expression E. coli cell and their recombinant protein expressions capacity and optimum length of time were studied after inducing with 1mM IPTG. One of the colonies was selected and used for mass production of recombinant protein and the produced protein was purified using Ni-NTA resin. The result indicated that the expression of recombinant CP was obtained only for cloned CTV-cp gene in TOPO vector within BLD21 star (DE3) E. coli cell and inducing protein for 4hours after addition of 1mM IPTG were given optimum amount of recombinant protein expression. The recombinant CTV-CP was highly bound to Ni-NTA resin and only eluted when washed with low pH buffer during the purification, and can be used for polyclonal antibodies production.Keywords: CTV, cp gene, SDS-PAG

Identification of GST Interacted Proteins under PRSV Infected Papaya Using Affinity Purification–mass Spectrometry

Glutathione S-transferases (GSTs) are multifunctional proteins involved in stress metabolism, which play major roles in biotic and abiotic stress responses. GSTs have found in all organisms which is a major phase II detoxification enzymes found in the cytosol. GSTs regulate peroxidase and isomerase activities, they protect cells against H(2)O(2)-induced cell death. Papaya rigsport virus (PRSV) is one of main biotic agent that cause damage in papaya. The disease symptoms are mosaic, chrolosis, ring spot and stunt, all the symptoms are the early stage of cell death. The main point of this research to investigate protein interaction of the PRSV interacted plant proteins and GSTs recombinant protein using the classical Affinity-purification-mass spectrometry (AP-MS) approache. The GST protein was heterologous expressed in E.coli system and the pull down assay was applied to expore the protein interacted complexs after that the protein complexs were indentified by LC-MS/MS. CTC1, Protein CCA1 isoform X1, Tetratricopeptide repeat (TPR)-like superfamily protein, PHD finger protein ALFIN-LIKE 9 and Fructose-bisphosphate aldolase-lysine N-methyltransferase proteins were identified. The interacted five proteins were predicted the protein network by STITCH program, the result show they associated with oxidative stress response mechanism. However, this is the basic intensive information that could develop to manufacture PRSV resistance variety in the future

Genetic Variation of Coleosporium plumeriae from Different Provinces in Thailand

Plumeria rust samples were collected from five provinces in Thailand, including Bangkok, Nakhon Pathom, Rayong, Chonburi and Yala. All five isolates produced the uredial stage but only the isolates from Bangkok and Yala also underwent the telial and basidial stages. The morphological characteristics of all three stages present in the life cycle of the isolates were studied under stereo, compound and electron microscopes. Ribosomal DNA (rDNA) sequences at 28S and ITS (internal transcribed spacer) regions were analyzed with those in the GenBank database by Nucleotide BLAST and phylogenetic analyses. Coleosporium plumeriae was identified as the causal agent of plumeria rust by structure morphology and rDNA sequences that revealed genetic variation of the fungus as well. In general, there were significant differences in the morphological characteristics of uredospores, teliospores and basidia among the isolates. However, the variation of spore morphology was not related to the sampling locations. According to the phylogenetic analysis of 28S rDNA sequences, the UPGMA tree grouped all C. plumeriae from Thailand and foreign countries in the same clade as they shared identical sequences. On the other hand, the UPGMA tree inferred from ITS rDNA sequence data detected genetic variation of the isolate from Chonburi and separated it into the distinct tree branch. In this study, structure morphology and ITS rDNA were suitable genetic markers for both interspecific and intraspecific taxonomy of C. plumeriae

A Chromosome-Scale Genome Assembly of Mitragyna speciosa (Kratom) and the Assessment of Its Genetic Diversity in Thailand

Mitragyna speciosa (Kratom) is a tropical narcotic plant native to Southeast Asia with unique pharmacological properties. Here, we report the first chromosome-scale assembly of the M. speciosa genome. We employed PacBio sequencing to obtain a preliminary assembly, which was subsequently scaffolded using the chromatin contact mapping technique (Hi-C) into 22 pseudomolecules. The final assembly was 692 Mb with a scaffold N50 of 26 Mb. We annotated a total of 39,708 protein-coding genes, and our gene predictions recovered 98.4% of the highly conserved orthologs based on the BUSCO analysis. The phylogenetic analysis revealed that M. speciosa diverged from the last common ancestors of Coffea arabica and Coffea canephora approximately 47.6 million years ago. Our analysis of the sequence divergence at fourfold-degenerate sites from orthologous gene pairs provided evidence supporting a genome-wide duplication in M. speciosa, agreeing with the report that members of the genus Mitragyna are tetraploid. The STRUCTURE and principal component analyses demonstrated that the 85 M. speciosa accessions included in this study were an admixture of two subpopulations. The availability of our high-quality chromosome-level genome assembly and the transcriptomic resources will be useful for future studies on the alkaloid biosynthesis pathway, as well as comparative phylogenetic studies in Mitragyna and related species