Cloning And Bioinformatics Analysis Of P25, P27 And P20 Genes Of Citrus Tristeza Virus

Citrus tristeza virus (CTV) is a causal agent of the most economically important diseases of citrus, distributing in the citrus production regions all over the world and threatening the citrus in- dustry seriously. CTV, the largest plant RNA virus, belongs to the genus Closterovirus, Closterov- iridae family, dispersed to new areas mainly by infected nursery tress and budwoods, and then locally spread by aphids. The brown citrus aphid (Toxoptera citricida)is the most efficient CTV vector. CTV virions of around 2000×11nm are filamentous and contain a single-stranded, positive sense genomic RNA(gRNA) molecule with 19,296nt possessing 12 open reading frames (ORF1a,ORF1b,ORF2～ORF11) including ORF7 coding major coat protein p25, ORF6 coding minor coat protein p27, and ORF 10 coding hydrophobin protein p20 which is a major component of fo- rm less inclusion of citrus infected by CTV. p20, p25 and p27 might play an important role in tr- ansmitssion of CTV by Toxo- tera citricida and have been considered to be related to pathogens- is of CTV. In this study, p25, p27 and p20 were cloned and their bioinformatics analysis has be- en addressed .The following results have been achived.1.Cloning of p25, p27and p20The rough extraction of CT11 nucleic acid was used as the template in the RT-PCR amplification purification of the complete ORF of p25, p27 and p20, and then the purified products were ligased with pTA-2 vector transformed into E.coli DH5a, selected through Amp resistance, and identified by bacterial colony PCR and EcoR I enzyme digestion. As a result, three reconstruction vectors named CTV-p25, CTV-p27 and CTV-p20 were constructed.2.Analysis of the nucleotide sequence of p25,p27 and p20The nucleotide sequences of p25,p27,p20 were analyzed with the DNAStar software.The complete ORF of p25 and p27 coded two coat proteins contained 672 and 723 nucleic acids, respectively. The content of base A in the two nucleic acids was the highest at the percentage of 28.87% and 31.12%, respectively, while base C was the lowest at 18.60% and 17.98%, respectively. The complete ORF of p20 possessed 549 nt, G, A and C were 160,139, 136, and 114 from high to low with frequency of 29.14%, 25.23 %, 24.77 % and 20.77%, respectively.3.Clustering analysis of p25, p27and p20Three genes of CT11 and 9 CTV isolates in GenBank which were sequenced completely were analyzed by clustering nucleotide and amino acids.In the nucleotide phylogenetic trees of p25 of CT11, and of VT, NuagA, SY568 and T318A aggregated in one branch,CT11 and T318A which comes from Mexico severe isolate aggregated in one branch of p27,T318A,NuagA and CT11 aggregated in one branch of p20.In the amino acids phylogenetic trees of p25,CT11 and SY568 aggregated in one branch.Similarity to the nucleotide phylogenetic trees of p27 and p20 their amino acids phylogenetic trees nearly reflect the same type of relationship to different isolates.4.Analysis of amino acids components and isoelectric pointsOf 223 amino acid encoded by p25, 31 were alkaline, 30 were acidic, 73 were hydrophobic amino acids, 61 were polar amino acids. They carried positive 1.329 charges at pH 7.0 and isoelectric point was 7.831. Of 241 amino acids encoded by p27, 35 were alkaline, 34 were acidic, 74 were hydrophobic and 63 were polar. They carried negative 1.475 charges at pH 7.0 and isolectric point was 7.753. Of 182 amino acids encoded by p20, 21 were alkaline, 22 were acidic, 68 were hydrophobic and 45 were polar. They carried negative 0.061 charges at pH 7.0 and isolectric point was 6.979.5.Prediction of the secondary structures of p25, p27 and p20The majority of the secondary structures of two coat proteins are random coil. The majority of the secondary structures of p20-encoded protein isα-helix.6.Transmembrane analysis of p25, p27, p20-encoded proteinsThe amino acid sequence of p25 encoding protein from 170 to 191 forms a helix from inside to outside and an outside to inside transmembrane helix from 170-194. The amino acid sequences of p27-encoded protein from 170 to 191 and of p27-encoded protein form a transmembrane helix from 17 to 33. Three transmembrane helixes of p20-encoded protein from inside to outside were found within the amino acid sequence from 96 to 117, 154 to170, and 163 to 182, respectively. In addition, one helix of the amino acid sequence from outside to inside was found from 96 to 116.