Cloning And Functional Analysis Of AcCOP10 And AcCDF2 Genes In Onion (Allium Cepa L.)

Onion?Allium cepa L.?belongs to Allium,is a typical biannual plant.Under the suitable condition,the bulbs are formed in the first year,after the winter storage of vernalization and under the suitable photoperiod condition,the plant undergoes flowering and fruiting to complete the plant life cycle in the second year.The regulation of the photoperiod pathway involves many regulatory elements,such as photosensitive pigment?PHY A,PHY B?,cryptoanthocyanin?Cry C?,clock rhythm element?CDF?and signal output element?CO?.In the photoperiod pathway,light receptors?such as PHY A,PHY B and Cry C?receive light signals and form a cascade of intracellular second messenger systems to regulate light morphogenesis.It has shown that COP gene acts as a suppressor of photomorphogenesis in the model plants and plays a central role in many light-mediated development processes in Arabidopsis thaliana,including the flowering and the control of the hypocotyl growth.The abundance and activity of COP are essential to the plants,and acts as a central inhibitory factor of the CO-FT flowering regulation pathway.The clock regulation pathway coordinates many developmental processes of photoperiod and temperature,and the most important element is CDF-FKF1.CDF gene acts as an endogenous rhythm gene and plays an important role in the regulation of photoperiod and temperature.Its effect on plants not only regulates flowering and hypocotyl elongation,but also participates in many processes such as cold tolerance and drought tolerance.In the photoperiod pathway,COP and CDF genes can inhibit circadian rhythms and the transcription levels of the photoperiod signal of CO-FT regulatory elementsand to inhibit the flowering of plants.In the life cycle of plants,they participate in multiple ways.In this study,COP and CDF genes were cloned.To study its expression patterns,we constructed the plant expression vector and identified the function by phenotypic changes of transgenic plants.Analyzed the changes of gene expression in the transgenic Arabidopsis thaliana plants.The functions of COP10 and CDF2 genes were clarified,and the flowering network of onion was further improved.The experimental results are as follows:?1?According to the results of transcriptome sequencing,primers were designed.AcCOP10?KY419117?had a full length of 531 bp,which was predicted to encode 176 amino acids.AcCDF2?KY406741?was 852 bp in length,presumably encoded 283 amino acids.The results of software analysis showed that the identity of COP10 was 68.88%,and there was a continuous sequence of WD40.The identity of CDF was 52.20%,which had a highly conserved C2-C2 DOF domain.?2?Phylogenetic tree analysis showed that AcCOP10 and Elaeis guineensisare were in the same branch,AcCDF2 and Dendrobium catenatum were close to each other.Several typical monocotyledon and dicotyledonous plants clustered in different branches,which might be due to the diversity of biological processes over long time,resulted in the evolution of CDFs adapted to its own changes,resulted in specific differentiation.But the similar zinc finger domains were retained.?3?The temporal and spatial expression of AcCOP10 and AcCDF2 was detected by fluorescence quantitative PCR.The results showed that the expression of AcCOP10 was higher in the leaf sheath without bolting than in the flower in the blooming stage after bolting.The expression of AcCDF2 was the highest in the young leaves without bolting,followed by the higher expression in the blooming stage after bolting.Except for this,the expression level in other parts was very low.?4?Two genes over-expression vectors p AcCOP10-OX,p AcCDF2-OX and RNAi vectors p AcCOP10-RNAi were constructed.The recombinant plasmid wasintroduced into A.tumefaciens LBA4404 using a freeze-thaw method.After that,wild type Arabidopsis thaliana was transformed with inflorescence infection.Compared with wild type lines,the number of rosette leaves was increased,plant growth was slightly dwarfed and flowering time was delayed in the two over-expression lines.p AcCOP10-RNAi plants showed earlier flowering and more tillers.?5?The over-expression vector of AcCOP10 was transformed into cop10 mutant?SALK0889930?.The flowering stage of the transformed plant was severely inhibited and insensitive to photoperiod.So it was difficult to sense light signal to regulate the transformation from vegetative growth to reproductive growth.In order to determine whether the phenotypic changes in late flowering of transgenic plants were due to clock-controlled flowering time genes,we analyzed the expression of CO and FT in transgenic plants.Compared with wild type and mutant plants,the expression levels of CO and FT in cop10 mutant were significantly higher than WT,which led to precocious flowering of mutant plants.The expression of CO in transgenic mutant was significantly lower than mutant,and it was also significantly lower than wild type,while the expression of FT in transgenic mutant was significantly lower than mutant,but there was no significant difference between transgenic mutant and wild type.These results suggested that AcCOP10 could recover the early flower phenomenon caused by cop10 mutation,which had the same function as At COP10 gene in regulating the downstream photoperiod response gene,which in turn regulated the flowering of Arabidopsis thaliana.The expression of CO in AcCOP10-ox-WT line was lower than WT,but the expression level of FT was only slightly lower than WT,indicated that AcCOP10 could cause the late flower of Arabidopsis thaliana,which was achieved by down-regulated the expression of CO.And it may not have passed through FT.In order to study the regulatory pathway of COP10,the m RNA level of FLC,vernalization mutant and non-vernalization mutant were analyzed and it was found that the expression of FLC in the mutant was significantly inhibited compared with WT.These results suggested that COP10 might be involved in vernalization and flowering through regulating the expression of FLC.AcCDF2 over-expression vector was transformed into cdf1 mutant?CS476144?and cdf5 mutant?SALK₀44252C?respectively.The transgenic plants recovered the bad phenotype of the mutant plants,improved the adaptability to the environment,prolonged the l ife cycle and delayed the flowering time,which indicated that AcCDF2 was involved in the regulation of biological clock related pathways.AcCDF2 might play a unique role in the transition of flowering plants,and the expression of CO and FT in transgenic plants was inhibited.These results suggested that AcCDF2 might act as a key negative regulator to produce a precise mechanism for regulating the expression of CO-FT.?6?In order to verify the role of AcCOP10 and AcCDF2 in flowering regulation,two genes over-expression vectors were transformed into Arabidopsis thaliana gi mutant?CS181?.Compared with gi mutant plants,the expression levels of CO and FT in AcCOP10-ox-gi and AcCDF2-ox-gi lines were significantly lower than wild plants.In combination with the results of Arabidopsis thaliana studies,it was confirmed that AcCDF2 was downstream of GI,and that AcCOP10 transgenic gi mutant had late flower phenotype,but it was obviously weaker than that of cop10 mutant,so it was speculated that AcCOP10 and GI could regulate flowering of plants in parallel.?7?The transgenic vector AcCDF2-OX,WT and cdf5 mutants were treated with drought stress.It was found that the plants of WT and AcCDF2-ox-cdf5 showed similar symptoms of severe water loss and severe wilting,but the survival rate was higher than WT.The cdf5 mutant died completely,the transgenic mutant slightly restored the tolerance of the functional deletion mutant and increased the survival rate of the plant.The transgenic plants of AcCDF2-ox-gi and gi mutant were less affected and maintained healthy green leaves.The survival rate of AcCDF2-ox-gi was higher and only a few of the leaves strain had yellowed wilting.The Gols2 was modulated in different degrees in transgenic plants,which indicated that AcCDF2 promoted the expression of Gols2,which might be used as an activator of drought stress pathway and improve the drought resistance ability of plants.?8?In order to verify whether AcCDF2 responded to freezing stress,it was found that cdf5 mutant plants was also significantly damaged after cold acclimation or non-acclimation,showing a lower freezing resistance.Compared with non-acclimated WT plants,AcCDF2-ox-gi,AcCDF2-ox-WT plants showed higher freezing resistance and higher survival rate.AcCDF2-ox-cdf5 plants recovered the freezing resistance phenotype of the mutants.Increased adaptability to extreme temperature environments.In addition,the cold tolerance of AcCDF2-ox-gi,AcCDF2-ox-WT were still stronger after acclimation at low temperature.The cold tolerance of AcCDF2-ox-gi plants was stronger than other transgenic lines,and the ada ptability of AcCDF2-ox-cdf5 was weaker than that of WT.The m RNA level of the cold-resistant central gene CBF1 was up-regulated in transgenic lines,suggested that AcCDF2 might be the upstream activator in the cold-stress pathway,directly or indirectly acted on the expression of the target genes regulated by the stress.?9?After dark treatment,WT showed small cotyledon and moderate Hypocotyl length;cop10 mutant showed strong hypocotyls inhibition phenotype and cotyledon opening.The Hypocotyl length of AcCOP10-ox-cop10 line was similar to WT,but the development of cotyledon was still slightly larger than WT;The phenotypes of AcCOP10-ox-gi and AcCOP10-ox-WT were similar,showing that cotyledon expansion were smaller and Hypocotyl elongation were signifi cant.These results suggested that AcCOP10 could promote Hypocotyl elongation in dark by different mechanisms independent of light.The expression of Hy5 in transgenic plants decreased to some extent,and the expression of Hy5 in cop10 mutants increased significantly.These results suggested that AcCOP10 might promote the elongation of hypocotyls by degrading the expression of Hy5.