Cloning And Functional Analysis Of Cotton Nodulin-like Gene

Nodulin gene is the gene that specific express in host plant during symbiotic nitrogen fixation of rhizobium and legume, participate in regulation of nodule growth,development and formation.It is significance in nodulation and nitrogen fixation.The function include transmembrane transport of hydrone ,glycerine and formamide ; participating in cellular differentiation and cell wall regenerate; histogenesis and signal transduction and participating in response of osmosis stress.With the further study, people colone nodulin-like gene in non-legume plants which is homologous with nodulin gene of legume plants.Some of these genes are the same in the function with the nodulin gene of legume plants and some are not same erther in expression pattern or in function.All above indicated that the function of nodulin-like gene of non-legume plants in different plants is diversity.Cotton nodulin-like gene is cloned by the isocaudarners inverse PCR during the study of cotton reproductive development gene. It was named"nodulin-like"because whose amino acid sequence has the highest identity with Nodulin MtN21 family At1g09380 protein .The gene is located in reproductive organ such as cotton fiber and young buds by the GUS report gene through the prophase work of our lab.But the gene function is still unknown.For further study the function of nodulin-like gene.The study constructs cotton nodulin-like gene and gfp gene fused expression vector and transformates to onion epidermis to transient express indicated that the gene located on cellular membrane .It was supposed to participate in material transmembrane transportation as a membrane integral protein.At the same time the vector of the full-length promoteor of nodulin-like gene drives GUS expression is constructed and transformates to model plant Arabidopsis.The gene located on embryonic tissue (cotyledon, caulicle and radicle),leaf primordium,petal,chapiter and capillament in the transgene Arabidopsis. The result suggests that cotton nodulin-like gene may involve in gametophytic development.In addition the transgene plant induced by 15% PEG6000 made GUS expression higher than control indicate that the gene response to drought stress. For further prove whether cotton nodulin-like gene act in osmosis stress, we bought T-DNA inserted mutant of At1g09380 gene of Arabidopsis.The number is salk₁40423. One homozygous T-DNA mutant was identified by PCR and RT-PCR identified the mutant is null mutant. The phenotype of the mutants were observed at the stages of germination and postgermination.The results showed that it made no difference between the germination of mutant and wild type in salt.And in 1/2MS of 200 mmol/L NaCl culture 11 day the survival rate of wild type is 66.67% and the survival rate of mutant is only 27.45%. The death rate of mutant is 62.79% and wild type is 9.30% after drought stress indicate that At1g09380 mutants are sensitive to NaCl and drought suggested that the gene may participate in salt and drought stress- resistance process in Arabidopsis.Meanwhile we cloned cotton nodulin-like cDNA by RT-PCR.First the hydrophobicity and phosphorylation site of amino acid sequence are analysised.The result is that there are 9 transmembrane helixes and 11 presumed phosphorylation sites.It is supposed that the 207 and 209 Ser and 206 Thr which located in cytoplasm side may happen phosphorylation with high possibility according to the distribution characteristic of phosphorylation site of membrane integral protein.Then construct plant expression vector transformate Arabidopsis.We get 43 lines transgene Arabidopsis by resistance screening and PCR.We choose 3 lines for further experiment by analysis the transcription level.To analysis the influence to cotton nodulin-like transgene plant with ABA,we found that the germination rate of transgene lines are lower than wild type on the 1/2MS contained 0.25μΜ,0.50μΜ,0.75μΜand 1.00μΜABA respectively suggest that the transgene lines are sensitive to ABA.For example,the germination rate is 64%,26% and 68% of the three transgene lines respectively and the rate of wild type is 82% in 1/2MS contain 1.00μΜABA culture 72h. In the seeding growth experiment the transgene lines are weaker than wild type indicate that the transgene lines are sensitive to ABA too.In the salt plate experiment,the survival rate of transgene lines are 64.57%,76.90% and 67.77% respectively and the wild type is 23.7%. And in the soil drought experiment the survival rate of transgene lines are 65.9%,72.6% and 68.1% respectively and the wild type is 38.67%.Analysis the result of real-time PCR of involved in abiotic stress induced and response genes suggest that transgene plant up regulate the expression of some gene such as ANAC019,DREB2A,KIN1,RD29B and P5CS1 and down regulate some gene such as RD22. Under the condition of 200mmol/L NaCl and 15% PEG6000, transgene plant up regulate the expression of some gene such as DREB2A,KIN1,RD29B and P5CS1 and down regulate some gene such as ANAC019 and RD22.The results above further indicated that cotton nodulin-like gene production participate in the response of osmosis stress.The paper is the first one that identify code production of cotton nodulin-like gene is a membrane integral protein.And we first identify that the nodulin-like protein is involved in the response of osmosis stress,enhance the drought resistance and salt resistance of transgene Arabidopsis through genetic transformation and expression in Arabidopsis further indicate that the function of cotton nodulin-like gene is diversity .The work makes a foundation for the further study of nodulin-like gene and provids new gene rescource to adversity resistance gene engineering of plants.