Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe gramin...Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .展开更多
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 in...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.展开更多
文摘Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .
基金Funds are provided by Higher Education Commission(HEC),Pakistan to Dr.Bushra RashidPrincipal Investigator of the Project No.20-3692/NRPU/R&D/HEC/14 to complete this study。
文摘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.
