Effects Of Low Temperature Stress On Expression Of Part Key Gene In JA Signaling Pathway And JA Content Of Tobacco At Seeding Stage

Low temperature is the major form of non-biological adversity,seriously affecting the yield and quality of crop,restricting the development of agriculture in many areas.Tobacco is a crop that has a high economic value for its leaf,the low temperature at seedling stage could limit the growth,reduce the number of available leaves,and affect its yield and quality seriously after transplanting to field.Jasmonic acid is a plant hormone found in recent years,regulating the whole growth and development process of plants,and playing an important role in defense responsing to biological and abiotic stress.At present,the regulating effect of jasmonic acid in plants such as Arabidopsis thaliana,wheat and rice has been studied,as well as the jasmonic acid in tobacco,but the reporting about the jasmonic acid signaling pathway in response to low temperature in tobacco stress has rarely been seen.In this study,Yunyan 85 and Xingyan 1 were choosed as experimental material.When they had 6 leaves in the seedling stage,they were disposed with low temperature(12?)and room temperature(28?)for 10 days,then their stem tip were taken to detect with the tobacco gene expression profile 4×44K chip provided by Agilent Company.The key candidate gene NtMYC2 in JA acid signal pathway was screened out through the analysis of GO and KEGG,and NtNINJA gene in JA acid signaling pathway was cloned.The dynamic expression differences of NtAOC,NtAOS,NtCOI1,NtJAZ1,NtLOX,NtMYC2 and NtNINJA were also analyzed through real-time quantitative PCR.Besides,the change of the JA of tobacco under low temperature and normal temperature was measured to analyze the relationship between low temperature stress of tobacco at seedling stage and JA signal pathway.The main results of this study were as follows:1.After detecting the gene expression profile of tobacco stem tip for 10 days under low temperature stress,two varieties had more up-regulated or down–regulated differentially expressed genes under the low temperature of 12? when compared with 28? stress.Compared with the Yunyan 85,up-regulated genes were far more than down-regulated in Xingyan 1 under the low temperature treatment.Through the analysis of GO and KEGG,three differentially expressed genes which were related to plant hormone signaling pathway called NtBRI1?NtPR1 and NtMYC2 were screened out.Besides,the expression of NtMYC2 in the stem tip of two cultivars was up-regulated at low temperature stress.2.According to the homologous conserved sequence of genes of the tobacco ninja-family protein in the NCBI database,special primers were designed,and the protein gene NtNINJA which interacts with JAZ in tobacco was cloned.The result of bioinformatics analysis showed that the CDS of this gene was 945 bp long and encoded 315 amino acids.And its protein was hydrophobic,non-signal peptide and transmembrane region by predicting,N-terminal had a kinase domain EAR,C-terminus had a TPL binding domain,which interacted with TPL-associated protein to help JAZ protein inhibit transduction of JA signal.It also had serine,threonine and glutamate receptor protein knaseactivity by Phosphorylation sites predicting.The genetic relationship betwen NtNNJA and gene sequence of ninja-family protein of Nicotiana tomentosiformis,which were in the same evolutionary branch,was the closest,and it had a close genetic relationship with pepper,potato,eggplant,tomato which also belonged to solanaceae.3.The results of quantitatively analysis of NtAOC,NtAOS,NtCOI1,NtJAZ1,NtLOX,NtMYC2 and NtNINJA genes showed that the expression level of NtMYC2 in both cultivars were higher than those in normal temperature control,it was consistent with the results of gene chip.At the same time,the expression level of NtMYC2 in Xingyan 1 was higher than that in Yunyan 85.In addition to NtJAZ1,the relative expression of the remaining genes under low temperature treatment were higher than normal temperature in these two cultivars,it indicated that low temperature stress enhanced their transcriptional activity,further induced JA signaling pathway response to low temperature stress.4.The jasmonic acid content of tobacco was measured under low temperature by Plant jasmonic acid(JA)ELISA Kit.The results showed that the JA content increased rapidly after the start of low temperature duress in these two cultivars and gradually decreased at the end of treatment.The JA content in most time points was higher except the 24 st day.This meaned that low temperature stress enhanced tobacco JA biosynthesis to improve its ability to cope with low temperature duress.And the total amount of JA reached the maximum at the 10 th day when the treatment was ended on Yunyan 85 and Xingyan 1,were 1869.566 and 2038.475 pmol/L respectively.The differences between them were 507.075 and 963.762,and the growth rates were 38.1% and 89.7% respectively,which indicated that the change of JA content in leaves of Xingyan 1 was more obvious under low temperature stress.In a word,the conclusion of this study was that low temperature stress could enhance the expression of key genes in JA signal pathway of Yunyan 85 and Xingyan 1,inducing a large number of JA synthesis,and then affected the mechanism of tobacco response to low temperature,produced the corresponding biological effects.In addition,Xingyan 1 in the JA signal pathway was more sensitive to the low temperature stress.By analyzing the expression of key genes in JA signal pathway and the changes of jasmonic acid content of tobacco under the low temperature stress,the foundation for further research was laid,such as how to improve plant resistance to stress and reduce low temperature damage at the seedling stage.