Function Analysis Of Four E3 Ubiquitin Ligase Encoding Genes From Caragana Korshinskii And C. Intermediaia

Caragana korshinskii and C.intermedia are important sand-fixing shrubs in northwest arid area with drought,cold and saline-alkali tolerance.Excellent stress resistant genes screened from it could provide good candidate genes for genetic engineering of forest and forage species.E3 ubiquitin ligases play a crucial role in regulating plant growth,development and stress response.In this study,EST sequences of three RING type and one U-box type E3 ubiquitin ligase encoding genes were isolated from dehydration-induced suppressive subtractive hybridizations?SSH?cDNA library of Caragana korshinskii and the transcriptome of C.intermedia.These four genes were cloned and its functions were analyzed.The detailed results are as follows:1.The expression pattern of three E3 ubiquitin ligase encoding genes in C.korshinskii was analyzed by qRT-PCR.They expressed in different organs of C.korshinskii and induced by ABA,NaCl and dehydration.2.The three CkE3 genes were isolated,and was named CkRNF185,CkRNF5,and CkRNF111 respectively.Sequence analysis shows that they encode 232,230 and 380 amino acids,respectively.Protein domain analysis indicates that all the three proteins contained classical RING domain.CkRNF185 and CkRNF5 belong to C3HC4 type,while CkRN111 belongs to C3H2C3 type.3.The prokaryotic expression vector was constructed to purify the three proteins,and the enzyme activity was detected by in vitro ubiquitination assay.The results showed that all three proteins had E3 ubiquitin ligase activity.4.Three E3 ubiquitin ligase genes were overexpressed in Arabidopsis thaliana,and the transgenitic plants were generated for analyzed.The results showed that overexpressing CkRNF185 in Arabidopsis reduced the tolerance to osmotic stress,drought and salt stress.Overexpression of CkRNF5 resulted in insensitive phenotypes to salt and osmotic stress during seed germination.Overexpression of CkRNF111 promotes early leaf senescence in transgenic Arabidopsis.5.Label-free protein quantitative method was used to investigate the mechanism of leaf senescence when CkRNF111 was overexpressed in Arabidopsis.A total of 85 proteins significantly differentially expressed,based on a 2-fold change,were detected by analyzing the differentially expressed proteins in the overexpressed lines and the control.Among which 65 proteins were up-accumulated,20 proteins were down-accumulated.KEGG enrichment results show that the major metabolic pathways involved in these proteins are starch and sucrose metabolism and phenylpropionic acid synthesis signaling pathways.GO enrichment results show that major biological functions include defense response,leaf senescence,stress response,stimulus response and macromolecular metabolism.Because the differentially expressed proteins are mainly involved in the starch and sucrose metabolic pathways,we speculated that it might be the sugar signal caused leaf senescence.However,many of the differentially expressed proteins are pathogenesis related and involved in defense responses.6.In addition,a homolog of AtPUB22?NP₁90813.1?,which showed 58%identity,was isolated from the transcriptome of C.intermedia and was named CiPUB22.The expression level of CiPUB22 was induced by ABA,NaCl and dehydration treatment,which was checked by qRT-PCR.Overexpression of CiPUB22 decreased tolerance of the transgenic Arabidopsis to salt and osmotic treatments during seed germination,and increased their sensitivity to ABA.