Functional Analysis Of PsARRO-1 In Adventitious Root Development Of Paeonia Suffruticosa

Adventitious rooting related oxygenase(ARRO-1)gene was specifically expressed in early adventitious rooting,and considered as one of the molecular markers of adventitious rooting.In this study,field seedlings and plantlets in vitroof Paeonia ostii ‘Fengdanbai’ were used to quantitatively analyze the expression of this gene in different parts,and in different development stages,namely pre-germination of root primordia,root primordium germination,root primordium protuberance androot growth,of adventitious rootingby q RT-PCR.Based on the full length of PsARRO-1(KJ620008),a fluorescent vector was constructed and subcellular localization of the gene was observed by laser confocal microscopy.Constructed the overexpression and knockout vector of PsARRO-1,andtransformed to Arabidopsis thalianaby Agrobacterium-mediated genetic transformation system,then identifiedthe transformed plants preliminarily.The main results were as follows:1.Analysis of relative expression of PsARRO-1The relative expression of PsARRO-1 in different parts of field seedlingsand plantlets in vitro of Paeonia ostii‘Fengdanbai’was analyzed by q RT-PCR.The results showed that PsARRO-1 was expressed in roots,stems and leaves,but the expression level was different.In plantlets in vitro,the expression of stem and leaf had little difference,and the trend was relatively stable.Although the expression of leaf was slightly higher than that of stem,the peak value of stem was slightly higher than that of leaf.The expression level of root increased sharply in 0-5 days,then decreased steadily after the peak value reached in 10 days,and second peak appeared in 35 days,which was 2-3 times as much as that of stem and leaf.In field seedlings,the expression of stem and leaf showed similar trend,peaking at the third and fourth sampling respectively,and the expression of stem was higher than that of leaf.The expression of roots was about twice as much as that of stems and leaves,and reached the maximum value at the fourth sampling,but then increased and decreased greatly,with multiple peaks and no obvious change rule.It is speculated that the complexity of root development,which leads to the non-synchronization of root development degree,thus causing greater errors in experimental results.On this basis,according to section observation,root primordia wasn’t found in new roots with just germinating length of2-3 cm,and obvious phenotypes of root primordia germination were found in new roots with root length of 5 cm and no protrusion.Therefore,according to the phenotypes of root length,protrusion,theadventitious root stage was divided into four stagesfor sampling,namelypre-germination stage(2-3 cm),root primordia germination stage(≥5 cm),root primordia,and root primordial breakthrough epidermisstage.Real-time quantitative fluorescence analysis showed that the expression of PsARRO-1 was the highest at the germination stage of root primordium,which was four times higher than that at the stage ofpre-germination,and twice as much as that at the protrusion and growth stage of root primordium.Therefore,it can be concluded that the gene is specifically expressed in adventitious root formation and plays a key role in root primordium germination.2.Localization analysis of PsARRO-1Constructed the fluorescent vector of PsARRO-1.According to the full-length sequence of PsARRO-1,the specific primers with the restriction sites of Pst I and Kpn I were designed.Digestedthe target gene and p Super1300-GFP vector plasmid by Pst I and Kpn I,thentransferredthe fluorescent vector into Agrobacterium tumefaciens GV3101,injected tobacco leavesand cultured for 48-72 hours.Laser confocal microscopy showed that the gene located in the plasma membrane.3.Construction of overexpression and knockout vectorConstructed the overexpression vector.The specific primers with Kpn I and Bam HI digestion sites were designed according to the full-length sequence of PsARRO-1.According to the vector construction scheme,digested the target gene and the over-expressed p CAMBIA1300 s plasmid by Kpn I and Bam HI,and linked the target fragment to form theoverexpression vector: 35s-PsARRO-1.Constructed the knockout vector.According to the sequence of PsARRO-1,the target was designed and inserted into the psg R-Cas9-At vector skeleton by Bbs I site to form an intermediate vector: PsARRO-1-Med-Cas9,thendigested the expression vector p CAMBIA1300 and PsARRO-1-Med-Cas9 by Hind III and Kpn I,and linked the target fragment to form the knockout vector: PsARRO-1-Cas9.The constructed overexpression and knockout vectors were transferred into LBA4404 by freeze-thaw method.4.Transformation of Arabidopsis thaliana by Agrobacterium-mediated inflorescence infectionThe overexpression and knockout vectors of PsARRO-1 were transferred into Arabidopsis thaliana by Agrobacterium-mediated inflorescence infection.After preliminary screening,hygromycin-resistant transformants were obtained.The results of PCR detection showed that the overexpression and knockout vectors were successfully transferred into Arabidopsis thaliana.By comparing the germination of transgenic T0 seeds with that of wild Arabidopsis thaliana,it was found that the root system of the plant with the knockout vector was obviously short.Because of the homology of the gene,the knockout expression of PsARRO-1 gene may inhibit the root development of Arabidopsis thaliana to some extent.Therefore,it is speculated that PsARRO-1 gene has a positive regulatory effect on root development.