ITRAQ-Based Proteomics Study Of Parthenocarpic Mechanism And Functional Analysis Of Auxin Receptor Genes In Cucumber(Cucumis Sativus L.)

Parthenocarpy is defined as successful fruit set in the absence of fertilization or other stimulation.In general,fruit set and development can be easily disturbed when pollination or fertilization is suppressed by sub-optimum growth conditions,such as low temperature or weak light intensity.However,parthenocarpic fruits could avoid yield loss caused by seed development and have a better texture,appearance and shelf life.Although cucumber is rich in parthenocarpic germplasm resources,the mechanism of parthenocarpy remains poorly understood,which restricted its application in germplasm enhancement.In previous studies,an excellent parthenocarpic cucumber cultivar,'EC1' was found,which showed environmentally stable parthenocarpy under different culture conditions.To improve the global understanding of parthenocarpy,we conducted a physiological and proteomic analysis of differently developed fruits based on 'EC1'.Besides,cucumber auxin receptor genes were cloned and functional analyzed.Main results of present research are as following:1.iTRAQ-based proteomics analysis of parthenocarpic mechanism in cucumberStudy on the growth traits of cucumber fruits indicated that the length and diameter of the parthenocarpic and pollinated fruits linearly increased from 0 to 6 dpa(days post-anthesis).The natural parthenocarpic(NP)fruits of 'EC1' and CPPU-induced parthenocarpic(CP)fruits of‘8419s-1' showed similar growth curves,wherein the fruit size was generally larger than the pollinated fruits of‘8419s-1'.In contrast,the growth of the unpollinated fruits of‘8419s-1' was blocked,and the length and diameter of the abortive fruits also decreased slightly.In addition,the endogenous hormones in the natural parthenocarpic fruits of 'EC1' were stable and relatively lower than those of the non-parthenocarpic fruits of '8419s-1',and the natural hormone-insensitive parthenocarpy of 'EC1' has broad hormone-inhibitor resistance.Based on the iTRAQ technique,683 fruit developmental proteins were identified from NP,CP,pollinated and unpollinated fruits.Forty-one parthenocarpy-specialized DEPs(differentially expressed proteins)were screened and divided into two distinctive groups:NP-specialized proteins and CP-specialized proteins.Furthermore,qRT-PCR and western blot analysis indicated that NP-specialized proteins showed hormone-or hormone-inhibitor insensitive expression patterns in both ovaries and seedlings.Physiological and proteomic comparisons indicated the presence of hormone-independent parthenocarpy and suppression of fruit abortion in cucumber.2.Cloning and functional analysis of auxin receptor genes in cucumberThere are two members in cucumber auxin receptor gene family,that is,CsTIRl and CsAFB2.qRT-PCR analysis revealed that these two genes may participate in the leaf architecture and fruit development processes.To further our understanding of their potential role in cucumber,two auxin receptor genes were overexpressed in tomato(Sonanum lycopersicum).The transgenic lines exhibited pleotropic phenotypes ranging from leaf abnormalities to seed germination and parthenocarpic fruit compared with the wild-type plants.The expression level of CsmiR393 was relatively low and steady during early stages of 'EC1' fruit development,while its expression level was dramatically up-regulated in'8419s-1' fruit at Odap.Semi-quantitative RT-PCR revealed that large amout of CsTIRl and CsAFB2 transcripts were cleaved in '8419s-1' fruit at Odpa.All these evidences indicated the existence of miR393-mediated mRNA cleavage of CsTIRl and CsAFB2,and highlighted the critical role of the miR393/TIRl homologs in mediating fruit set and development in cucumber.