Cloning And Functional Analysis Of Cotton E3 Ubiquitin Ligase Gene GhRING1-like

Drought is one of the most common environmental stress factors that influence plant growth and development,and has become the primary cause for reductions in crop yields.The eontradiction between the supply and demand of water resources and the ecological water shortage caused by irrigation in cotton production areas have become an important factor restricting the sustainable development of cotton production in China.Drought stress can lead to a series of physiological and biochemical responses such as stomatal closure,cell division suppression,and photosynthesis inhibition.When subjected to drought stress,plants can respond and adapt to the adverse condition by triggering a network of signaling events to maintain cellular function.The ubiquitination/26S proteasome pathway is a dominant protein turnover system that plays important roles in hormone regulation,embryogenesis,photomorphogenesis,circadian rhythms,floral development,senescence,and biotic and abiotic stress responses.In this study,the RENG-finger E3 ubiquitin ligase gene GhRLNGl-like was cloned from cotton and its gene function was studied.The specific results are as follows:1、Based on transcriptome data analysis,the GhRINGl-like gene up-regulated in drought stress was screened and molecularly cloned,Sequence analysis showed that GhRINGl-like consists of a 996 bp of open reading frame,which encodes a protein of 332 amino acids with a zine-ribbon domain at the N-terminus and a RING-H2(C3H2C3)domain at the C-terminus.The tissue expression characteristics of GhRING1-like and the expression patterns under different stress treatments were analyzed by qRT-PCR.The results showed that GhRING1-like was expressed in roots、stems、leaves、flowers、ovules and fibers of cotton,and the expression level was highest in leaves.GhRING1-like can respond to various stress treatments such as PEG,NaCl,ABA,and SA,but the temporal dynamic model of expression is slightly different.The GhRING1-like-GFP fusion protein was transiently expressed in tobacco leaves for subcellular localization analysis.The results showed that the GhRING1-like encoded product was located on the endoplasmic reticulum.2、VIGS silencing GhRING1-like cotton significantly reduced in relative water content and chlorophyll fluorescence parameter of Fv/Fm,while significantly increased in electrolyte leakage rate and malondialdehyde content compared with the wild type cotton under water deficit.In control,the GhRING1-like silenced plants were more severe than the controls after 12 days of natural drought.Transgeuic Arabidopsis thaliana was obtained using the bubble method.Overexpression of GhRING1-like significantly increased the tolerance of Arabidopsis germination and young seedlings to the osmotic stress derived from mannitol.Moreover,transgenic Arabidopsis at stage of middle to late vegetative growth showed decreased water loss rate and stomatal apertures coupled with 2.4-fold higher plants survival rate in comparison with WT under water deficit.3、The real-time quantitative PCR was used to analyze the Marker genes AtPLD,AtAREB1、AtERD15、AtRD29A and AtDREB in drought stress pathways.Under drought stress,the expression of ABA dependent drought inducing genes including AtAREB1,AtERD15 and AtRD29A was upregulated,but that of ABA independent drought stress-inducing genes of AtDREB and proline synthesis gene AtPLD was unaffected in the overexpressed GhRING1-like Arabidopsis.The pGBKT7-GhiING1-like bait vector was constructed and transferred into yeast.The GhRING1-like interaction protein was screened by the yeast two-hybrid system and 7 interaction proteins were screened.Two of them were found through the NCBI database.In summary,GhRING1-like can respond to a variety of abiotie stresses and play an important role in plant drought resistance.GhRING1-like mainly regulates the drought resistance of plants mainly relying on the ABA pathway.