Isolation And Functional Analysis Of A Protein Tyrosine Phosphatase Gene GhIBR5 From Cotton

Plants and other multicellular organisms exist obvious difference,plants can not elude harmful natural environment through their own movements and can only rely on highly sensitive response mechanisms to adapt to the changes of the environment.When plants are subjected to various stress injury from environment,a series of complex defensive responses are initiated to resist these stresses and to regulate the expression of related genes.The reversible phosphorylation of proteins are regulated by protein phosphatase(PP)and protein kinase(PK),this process play a key role in the process of defensive responses.As a regulatory protein,PP has been shown to be involved in stress-induced signal transduction in plants.Protein tyrosine phosphatases(PTPs)is a typical PP,recent studies have demonstrated that PTPs response to signaling pathways of adversity and control the growth of many physiological processes in plants.Cotton is a strategic commodity involved in the People's Livelihood,separating the stress resistance gene by applying the molecular biology,and cultivating stress resistance cotton species is an important goal of modern agricultural research.In this study,we used the(Gossypium hirsutum L.)(Lumian 22)as a material to isolate the IBR5 gene of the PTPs family and named GhIBR5.We have carried on the sequence analysis to this gene,the protein subcellular localization,the expression characteristic analysis and the biological function appraisal,the main research result are as follows:(1)The cDNA sequence of this gene was 1263 bp in length,including 816 bp open reading frame(ORF),182 bp 5' untranslated region and 265 bp 3' untranslated region.The ORF encodes a polypeptide containing 271 amino acid residues,which is predicted to have a molecular weight of 30.205 kDa and p I is 6.65.Analysis of homologous sequence alignment and protein cluster analysis showed that the gene we cloned is a typical PTPs gene.(2)Using the tobacco leaf to proceed on transient expression,we found that the GhIBR5 located in the cytoplasm and nucleus by subcellular localization analysis,so we speculated that the gene played a certain biological role in the nucleus and cytoplasm.(3)By analyzing the promoter sequence of GhIBR5 using PlantCARE and PLACE,we found a series of cis-acting elements in response to environmental stress.The experiment result of qRT-PCR and Western blot showed that the expression of GhIBR5 was up-regulated by low temperature(4?C),drought(15% PEG6000),mechanical injury(Wounding),methyl jasmonate(MeJA)and gibberellin(GA3).Under high salt,Ralstonia solanacearum(R.solanacearum),Rhizoctonia solani(R.Solani)and salicylic acid(SA),the expression of GhIBR5 was down-regulated.The above results suggest that GhIBR5 may respond to multiple processes of biotic or abiotic stresses.(4)We obtained the transgenic tobacco plants of GhIBR5.The results of biological function analysis showed that the overexpression of GhIBR5 gene reduced the resistance to drought at germination and seedling stage.In addition,the water loss analysis showed that GhIBR5 overexpressing plants increased the susceptibility to drought stress.(5)After the leaves were infected with R.solani,transgenic tobacco leaves had a larger lesion area,it indicates that transgenic tobacco reduced the resistance to R.solani.In addition,the virus-mediated gene silencing(VIGS)technique was used to obtain the silenced plants of GhIBR5.Through carries on the detection of diease-resistance,we found the silence of GhIBR5 improved the resistance for plants to R.solani.Thus,the GhIBR5 gene may respond to the pathogenic signal transduction pathway.(6)DAB staining was carried out after drought stress treatment for plants.It was found that the leaves of overexpressed plants had more ROS than WT plants,and the oxidative damage was more serious.There was less accumulation of ROS in plants that silenced the GhIBR5 gene.The enzymatic activitives of SOD,POD and CAT in transgenic tobaccos were more obvious after drought and R.solani treatments.It suggests that GhIBR5 may be involved in drought and pathogen stress responses in plants by regulating the level of ROS.(7)Through the yeast two-hybrid system,we identified Gh IBR5 and GhMPK3,GhMPK6,GhMPK11 or GhMPK12 interaction,which involves a variety of signal path and other complex processes remains to be further verified.