| Lotus belongs to Nymphaeaceae,and it is one kind of aquatic herb.In addition,lotus root is also a horticultural crops with high economic value in the world.The roots,stems and seeds of lotus root can be used as the daily diet,and while some organs are also valuable medicinal resources.The main root of lotus root is under developed,and the adventitious root plays a role in water and nutrients uptake,fixing and supporting the plant during plant growth.Therefore,it is necessary to explore the molecular mechanism on adventitious root formation of seedligs,which is helpful to uncover the regulation on lotus grwoth and development.The adventitious root formation of lotus root is affected by multiple factors,and among which,plant hormones excert an important role on the formation of adventitious roots.Auxin is firstly discovered among plant hormones,which is testified to involved in the regulation of adventitious root formation.Previous studies in our laboratory showed that auxin affected the formation of adventitious roots in lotus seedlings.Based on the data of previous experiments,this study preliminarily explored the regulatory mechanism of auxin on adventitious root formation of lotus seedlings from the microstructure,physiological and biochemical indicators and molecular levels.We obtained the following results:1.The development process of lotus adventitious roots was analyzed on morphology and microstructure after treatment with 10 μM IAA,and water treatment was used as the control.The results showed that the process of adventitious roots formation in the lotus seedlings was promoted by application of 10 μM IAA.Adventitous roots appeared on hypocotyls of seedlings treated with 10 μM IAA on the 4th day,and while six days were needed in the control plants.It was found that the primordium of adventitious root was formed and turned to elongated growth on the second day after treatment with auxin,and the cells of root primordium were observed on the fourth day in the control plants.2.After 10 μM IAA treatment of lotus root seedlings,we found that the activities of POD and SOD in the treated plants and control plants were firstly decreased,and then increased within 0-8 days,and the activities of POD and SOD in treated plants were higher than that of control plants;The activities of IAAO and PPO showed different trends between the treated plants and control plants.The treated plants had higher activities of IAAO and PPO than control plants.The starch content in treated plants was more than that of the control plants.The starch in these two types of plants was firsty decreased,and then increased from 0 to 4 days,and lower content in treated plants were found from 4 to 6 days after comparing with control plants.The content of soluble protein in treated plants was significant lower than that of control plants,and these content showed decrease tendency within observation period.Exogenous applicatin of IAA dramaticly decreased endogenous IAA and gibberellin content,and increased ABA content.3.RNA-seq technique was used to analyze the changes of gene expression after IAA(10μM and 150 μM)treatment.The results showed that there were 2431 differentially expressed genes in A/CK library including 1581 up-regulated genes and 850 down-regulated genes,and 1900 differentially expressed genes in B/CK library including 1183 up-regulated genes and 717 down-regulated genes;There were 1723 up-regulated genes and 1135 down-regulated genes in the C/CK library.Based on analysis of these differentially expressed genes,we found that the expression of NnARF17 and NnARF18 was significantly different between the treatment plants and the control plants,so two genes were used as candidate genes for further analysis.4.NnARF17 and NnARF18 were cloned from lotus by RT-PCR method.The results showed that the full-length NnARF17 and NnARF18 were 1800 bp and 2055 bp,which encoding 599 and 684 amino acid residues.NnARF17 has B3 and Auxin-resp domains,and NnARF 18 has B3,Auxin-resp and Aux-IAA domains.Three-dimensional space of NnARF 17 and NnARF 18 was constructed by homology modeling,and the results showed that NnARF 17 and NnARF18 belonged to typical auxin response factors.Phylogenetic tree analysis showed that NnARF17 was an isolated clade and lacked homologs,and while NnARF18 was closely related to BvARF18,CpARF18,ZjARF18,GhARF18,PtARF18 and JcARF18,so these genes were classified into the same clade.5.The expression patterns of NnARF17 and NnARF1 8 in treated plant with 20 g/L sucrose,10 μM IAA and 200 mg/L ethephon for six days were studied by qRT-PCR method,and βactin was used as the internal standard.The results showed that NnARF17 and NnARF18were induced by 20 g/L sucrose,10 μM IAA and 200 mg/L ethephon,and their expression patterns were similar.The mRNA level of NnARF 17 reached the maximum on the second day after treatment with the these solutions,and while NnARF18 reached the maximum transcriptional level on the sixth day.Further analysis showed that NnARF17 and NnARF18 had the characteristics of organ-special expression.NnARF17 showed more transcriptional level in fruit than other organs,and the lowest was found in flowers.The expression of NnARF18 was higher in roots than seeds,stem,leaf,and the lowest mRNA level was observed in flowers.6.The pCAMBIA1300-35S-EGFP-NnARF17 and pCAMBIA1300-35S-EGFP-NnAR F18 were successfully constructed,and then transformed into Arabidopsis thaliana by pollen tube pathway.We found that overexpression of NnARF17 and NnARF18 signifi cantly increased root length and root number in Arabidopsis plants than that of wild type plants,suggesing that NnARF17 and NnARF18 was involved in root developmen t in Arabidopsis plants.In conclusion,auxin affects the formation of adventitious root in lotus root,and NnARF17 and NnARF18 might be the key target genes in auxin signal transduction p athway to regulate the formation of lotus root. |
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