Cloning And Function Analysis Of Polyamine Oxidase GhPAOs Gene In Upland Cotton(Gossypium Hirsutum L.)

Object : Cotton is one of the most important economic crops, and the production of upland cotton（Gossypium hirsutum L.） accounted for about 90% all over the world. Planting area of cotton in Xinjiang province is becoming larger and larger. Low temperature, salinization and drought affect plant growth and development, endanger plants survival. Previous researches reported that the plants polyamine has close relationship with abiotic stress, and polyamine oxidase is a key enzyme of polyamine degradation,which produced H₂O₂, and H₂O₂ is closely related to plant growth and development, especially in the closely related with plant abiotic stress. Our study focused on the plant PAO gene cloning and functional analysis,which may provide important basis for the use of genetic engineering for plant genetic traits.Method:（1） The method of homologous sequences was used, combining with Raymond’s cotton genome information, and c DNA was set as a template to clone Gh PAO1/2/3 genes in upland cotton.（2） q RT-PCR analysis was explored for characteristics of different tissues and treatment conditions.（3） Gateway technology was used to build overexpressing p GWB17-Gh PAO12/3 expression vector, and transformed into Arabidopsis and cotton, respectively.（4） Determined the content of polyamine in wild-type Arabidopsis and p GWB17-Gh PAO1 plants by HPLC method.（5） DAB situ coloring was used for determination of H₂O₂ content in Wild-type and p GWB17-Gh PAO1 Arabidopsis.Results and Conclusion: 1. A homologous polyamine oxidase（PAO） gene was cloned through blasting At PAO1、At PAO2 and At PAO5 c DNA in Raymond’s cotton genome database. The new PAO was identified using RT-PCR and named as Gh PAO1 、 Gh PAO2 and Gh PAO3 in upland cotton. The full length of Gh PAO1、Gh PAO2 and Gh PAO3 c DNA was 1 751 bp、2 237 bp and 1 736 bp, including1 482 bp、1 473 bp and 1 530 bp open reading frame, encoding 493、490 and 506 amino acid residues, and the predicted protein molecular weight was 55.3709KD、54.405 k D and 56. 88 k D, isoelectric point was 5.31、5.69 and5.77, Encoding protein is hydrophilic protein. Clustering analysis is divided into three categories, Cotton Gh PAO1 gene, Tc PAO1, Vv PAO1 and At PAO1 together for a class, Cotton Gh PAO2 gene, Tc PAO2,Vv PAO2, At PAO2, At PAO3, Nt PAO2 and Md PAO2 together for a class, Cotton Gh PAO3 gene, Tc PAO5,Vv PAO5, and At PAO5 for category. Expression pattern of Gh PAO1、Gh PAO2 and Gh PAO3 was analyzed in different tissues and treatments using real-time PCR, The results showed that Gh PAO1 had a relatively high expression level in stems, Gh PAO2 had a relatively high expression level in flower petals and leaves,Gh PAO3 had a relatively high expression level in leaves. Gh PAOsexpression levels were significantly changes in different treatments. Expression level of Gh PAO1 reached the highest level at 1 h under ABA treatment and low temperature,The expressions of Gh PAO2 were quickly increased and reached the highest level at 1 h under low temperature or 20% PEG treatment, and the highest expression level was at 24 h under 200 mmol?L-1 Na Cl or 1 mmol ABA treatments. Expression levels of Gh PAO3 were significantly changed in different treatments. Expression level of Gh PAO3 reached the highest level at 6 h under low temperature and ABA treatment, whereas the highest expression level of Gh PAO3 occurred at 3 h and 24 h under Na Cl and PEG treatments, respectively. These results indicated that Gh PAO1、Gh PAO2 and Gh PAO3 was induced by abiotic stresses,and might play an important role in defending against abiotic stress in cotton.2. Transform p GWB17-GhPAO1 overexpressing vector into the model plant arabidopsis thaliana, Hyg filter to obtain pure fit and verified by PCR and QRT- PCR, all obtained positive transgenic plants, Choose the high amount of gene expression to Na Cl stress strain.（1） Determination of polyamines show transgenic lines of polyamines（PAs） reduced the total content put content increased, Spd and Spm content reduced.DAB chromogenic results show H₂O₂ content increased in transgenic lines. Therefore, we speculate that Gh PAO1 overexpressed genein Arabidopsis, may be involved in the degradation of Spd and Spm and produce more H₂O₂.（2）Exogenous polyamines and inhibitor treatment of wild-type and transgenic Arabidopsis showed, exogenous polyamines and PAO inhibitor（1,8-DO） promote Arabidopsis cotyledon green, Polyamine inhibitor（D-Arg） restrained cotyledon green in arabidopsis. Exogenous polyamines Gh PAO1 gene overexpression may cause an increase in the content of H₂O₂ by polyamine oxidation processing, thereby promoting green cotyledon.（3） Na Cl and adding exogenous polyamine treatment under Na Cl, Gh PAO1 transgenic arabidopsis green cotyledon rate is lower the wild type, Transgenic H₂O₂ content is higher the wild type, thus, H₂O₂ produced inhibitor green cotyledons in Arabidopsis. With 300 mmol Na Cl dealing with wild type and Gh PAO1 transgenic arabidopsis thaliana seedlings, the results show,wild-type arabidopsis leaves appear curly and water loss when Na Cl dealing with 36 h, even began to turn yellow, but transgenic line leaves is green peace and is less water loss. Using high pressure liquid chromatography（HPLC） analysis polyamine content in wild type and transgenic arabidopsis under high salinity, we found that the transgenic arabidopsis PAs content is lower the wild type.3. Constructed pGWB17- GhPAO1/2/3 plant expression vector, using the method of agrobacterium infect cotton hypocotyl transformation YZ-1, successive transfer culture and Kana screening for resistant plants.Extraction resistance of cotton genomic DNA for PCR verification, found that genetically modified for hypocotyledonary axis growth was significantly higher than that of no load growth.