Mechanism Of ThLEA3Overexpression In Phellodendron Amurense Rupr. Increase Osmotic Stress Tolerance

Late embryogenesis abundant proteins (LEA proteins) is a group of multifunctional adversity protein, which accumulating late in plant seeds embryogenesis. The results showed that LEA can be induced by drought and ABA stresses, and the expression level is positively relative to ABA content. Many studies indiated that LEA overexpression could improve the drought resisitant ability. However, up to now, less information on LEA functional moleculat mechanism and not perfect understanding of structure and function were known, also lots of controversy exsited. In this study, ThLEA3transgenic Phellodendron amurense Rupr. was used as research object, by the methods of physiological indexes change, stomatal closure degree, fluorescence molecular probe, SIET technology and Illumina high-throughout sequencing, change of NO, H2O2and ABA in stomatal closure process were investigated. We firstly researched the mechanism of rapidly stomatal closure under osmotic stress, and speculated the pobable molecular protective mechanism under this condition. The maining results are listed as following:(1) After7%PEG6000treated for10h, the leaves of non-transgenic plant apparently wilting with servious water loss, while ThLEA3transgenic plant still grown normally, indicatingThLEA3transgenic plant confer to osmotic tolerance. Different physiological indexes results showed that there is no differece of SOD, CAT activity between non-transgenic and transgenic plant under osmotic stress, while proline, soluble protein and MDA content in transgenic plant were higher than that in the control one.(2) SEM observation result indicate that stoma of transgenic plant closed, while stoma of non-transgenic plant was opened, even deformed under7%PEG6000, suggesting ThLEA3improved osmotic tolerance by rapidly stomatal closure.(3) Fluorescence probe detection results showed that, NO in transgenic P. amurense stoma was stronger than that in non-transgenic plant. But H2O2fluorescence result was opposite to NO. Meanwhile,Ca2+fluorescence in transgenic plant stoma was higher under ABA, H2O2, NO donror SNP and PEG6000treatment, also induced stomatal closure. SIET result showed that Ca2+was influxed, K+and H+were effluxed under PEG6000stress, finally, induced stomatal closure. But Ca2+、H+、K+in stoma of non-transgenic plant effluxed, resulting in gard cell damaged and not closed.(4) Content of endogenious ABA in non-transgenic plant leaves was higher than that in transgeni plant leaves with non-treatment. After7%PEG6000treatment, ABA in both ones greatly increased, however, ABA content in non-transgenic plant leaves was still higher than this in transgenic plant. (5) To further verify the expression of relative genes at trascrition level, transcriptome of non-and transgenic plant leaves were analyzed by Illumina high-throughout sequencing. The sequencing results indicated that ThLEA3overexpression changed expression level of30,494genes, among which,12263were up-regulated, and18231were down-regulated. Further analysis results showed that genes involve in ABA synthesis pathway presents the transition from up-rugulaion to down-regulation trend in transgenic plant compared with non-transgenic plant. In addition, the homologous genes of abscisic aldehyde oxidase, the last key step, involve in ABA synthesis were all down-regulated. Interestingly, all the gene relative to NO synthesis, such as NOS and NR were up-rugulated.