Identification,Isolation and Characterization of GaCyPI Gene in Gossypium arboreum under Cotton Leaf Curl Virus Disease Stress

Pakistan is facing the threat of Cotton Leaf Curl Virus(CLCuV)which is transmitted through whitefly to cotton crop.Molecular mechanism of leaf epicuticular wax protects the plants from different pathogens including insect attack and disease transmission.Objective of current study is the isolation and characterization of a wax related gene GaCyPI from Gossypium arboreum under CLCuV infection.A fragment of 475 bp was isolated from the total RNA and 3’and 5’RACE-PCR products were arranged by overlapping the extended sequences at both the ends.Deduced protein sequence of GaCyPI showed homology with Cyclophilin cis-trans isomerase gene of Gossypium ramondii and Gossypium barbadanse.Multiple sequence alignment also revealed homology among the coding sequences of same gene.GaCyPI protein comprised of 173 amino acids and ORF finder revealed the 69 bases upstream at 5’while 350 bp at 3’UTR.InterProScan revealed that it belongs to Cyclophilin-type peptidyl prolyl cis/trans isomerase(PPIase)family.Active sites are visible at specific amino acid positions and 3D structure was stable in Ramachandran plot.Prosa server showed protein residues have average 3D-1D score>=0.2 and Z-Score was−6.74.Phylogenetic analysis revealed that G.raimondii is the closest species that shares the same sequence.Hence,GaCyPI has strong role in plants’epicuticular wax and its genetic transformation may protect the cotton from whitefly which transmits CLCuV.

Genomic Epidemiology of SARS-CoV-2 in Pakistan

COVID-19 has swept globally and Pakistan is no exception.To investigate the initial introductions and transmissions of the SARS-CoV-2 in Pakistan,we performed the largest genomic epidemiology study of COVID-19 in Pakistan and generated 150 complete SARS-CoV-2 genome sequences from samples collected from March 16 to June 1,2020.We identified a total of 347 mutated positions,31 of which were over-represented in Pakistan.Meanwhile,we found over 1000 intra-host single-nucleotide variants(iSNVs).Several of them occurred concurrently,indicating possible interactions among them or coevolution.Some of the high-frequency iSNVs in Pakistan were not observed in the global population,suggesting strong purifying selections.The genomic epidemiology revealed five distinctive spreading clusters.The largest cluster consisted of 74 viruses which were derived from different geographic locations of Pakistan and formed a deep hierarchical structure,indicating an extensive and persistent nation-wide transmission of the virus that was probably attributed to a signature mutation(G8371T in ORF1ab)of this cluster.Furthermore,28 putative international introductions were identified,several of which are consistent with the epidemiological investigations.In all,this study has inferred the possible pathways of introductions and transmissions of SARS-CoV-2 in Pakistan,which could aid ongoing and future viral surveillance and COVID-19 control.