Functional Study Of The HECT-Type E3 Ubiquitin Ligase GhUPL7

Protein degradation plays an extremely important role in the cell life activities.The ubiquitin-proteasome system(UPS)is one of the major mechanisms of plant protein degradation,affecting almost the whole growth and development processes of plants,including photomorphogenesis,cell cycle,the ovule development,senescence and defense,environmental response and hormonal signal response.The ubiquitin-proteasome system is mainly composed of three enzymes: ubiquitin activating enzyme E1,ubiquitin conjugating enzyme E2,and ubiquitin protein ligase E3.UPL7 is a kind of HECT(Homologous to the E6-associated protein carboxyl terminus)ubiquitin ligase.The Arabidopsis HECT-like ligase have seven members: UPL1-UPL7.Among of them,UPL3 and UPL5 can regulate the plant growth and development through the ubiquitination pathway,indicating that HECT-type ligase participates in this pathway.In the past years,GhUPL7 was cloned from upland cotton(Gossypium hirsutum L.).To explore the function of HECT-like E3 ubiquitin ligase gene GhUPL7,suppression GhUPL7 transgenic cotton and ectopic-expression GhUPL7 in Arabidopsis thaliana are obtained by Agrobacterium-mediated transformation.The transgenic plants were analyzed.The main results are as followed:1.Analysis of the GhUPL7 gene expression pattern.The gene expression characteristics reflect its physiological function of the gene to a certain extent.The expression specificity of the tissues,organs and the analysis of its promoter showed that the expression of GhUPL7 gene is tissue-specific and has a high expression level in stamens and ovules.2.Down-regulation GhUPL7 gene affect the quality of cotton mature fiber.E3 ligase affects crop yield or not,the suppression GhUPL7 transgenic cotton seed yield traits including lint percentage,seed index,lint index,number of seeds per boll and boll weight were investigated.The results showed that when the gene wasdown-regulated,the content of lint percentage,seed index and lint index significantly increased,while the number of seeds per boll significantly decreased.It indicated that GhUPL7 down-regulated affects the ovule and fiber development,and the reduction in the number of seeds per boll,ultimately resulting in a decrease in the weight of the boll.The results of the cotton fiber length showed that the fiber of the transgenic material was not significantly different from the wild type.3.GhUPL7 affects post-translational modification of AILP1 protein.Based on the results of previous experiments,it was speculated that GhUPL7 may ubiquitinate GhAILP1 and the expression of GhUPL7 gene was consistent with the AILP1 gene expression.Therefore,the expression of AILP1 protein in GhUPL7 transgenic cotton and wild-type cotton was detected by western blot.The level of AILP1 protein in antisense GhUPL7 transgenic cotton was higher than that of wild type cotton.At the same time,the level of AILP1 protein in three A.thaliana plants lines heterologous expression GhUPL7 Arabidopsis,wild type and upl7 mutants was detected.The results showed the strongest signal in the upl7 mutant,followed by the signal in wild type,and the signal in the ectopic-expression GhUPL7 transgenic Arabidopsis plant was the weakest.This indicated that GhUPL7 protein affects post-translational modification of AILP1 protein.4.Heterologous expression of GhUPL7 gene leads to early bolting and increase the number of no.first-order rosette lateral branches formation.Wild-type and heterologous expression GhUPL7 Arabidopsis plants were simultaneously planted in nutrient mash to investigate.It was found that heterologous expression GhUPL7 in A.thaliana showed an earlier bolting and increased number of rosette lateral branches formation from rosette leaves than wild-type plants.It has been speculated that heterologous expression the GhUPL7 gene affects the no.first-order rosette lateral branches development in A.thaliana.5.GhUPL7 gene takes part in plant photomorphogenesis.Excessive expression of GhUPL7 gene causes whitening of cotton,the seedling suffered in changing to green shooting.It has been reported that E3 ligase COP1 is involved in plant photomorphogenesis and acts as a negative regulator of photomorphogenesis in dark conditions,interacting with HY5,leading to Arabidopsis hypocotyls elongation.Thus in order to explore whether GhUPL7 gene is involved inplant photomorphogenesis,we analyzed the effect of GhUPL7 gene on embryonic elongation of A.thaliana.The results showed that the hypocotyls of heterologous expression GhUPL7 A.thaliana plants under light conditions were significantly elongated and reached significant levels,but the mutants became shorter.Under dark conditions,heterologous expression of GhUPL7 was also observed the same change.Promoted the elongation of hypocotyls of A.thaliana,but the phenotype was obvious under no light conditions,and the hypocotyls of mutant plants became shorter.It has been that the GhUPL7 gene needs to degrade the positive regulatory factors of photomorphogenesis under the light condition.BBX20 and BBX25 are negative regulators of photomorphogenesis and promote the elongation of hypocotyls,whereas BBX22 is a positive regulator of photomorphogenesis and inhibits the elongation of hypocotyls.The expression levels of key transcription factors were detected in plant photomorphogenesis pathway.The results showed that compared with wild type,BBX20 and BBX25 transcription factors were highly expressed in heterologous expression GhUPL7 Arabidopsis plants,but expression was inhibited in mutants,BBX22 was reversed in the mutants.The type is high,but the expression level is low in heterologous expression GhUPL7 plants.It imply that GhUPL7 is a negative regulator of photomorphogenesis,but its target gene has not been found.