Cloning And Functional Analysis Of IiYUCCA6 Gene From Isatis Indigotica Fort.

Isatis indigotica Fort.,a traditional Chinese herbal medicine,contains many compounds,which have various pharmacological activities such as anti-virus,anti-tumor and anti-inflammatory.IAA,as an endogenous auxin,plays an important role in plant growth and development.At the same time,many studies have found that transgenic plants overexpressing YUCCA family genes have showed high endogenous IAA content and exhibited high salt,drought and low temperature stress tolerance in recent years.Furthermore,some research suggested that the above transgenic lines could delay the senescence of plant under dark stress conditions.Up to now,it has not been found any research about the function of YUCCA gene family in Isatis indigotica Fort.Therefore,based on the transcriptome database of Isatis indigotica Fort.,the appropriate reference genes were selected from 10 candidate reference genes for qRT-PCR technology under different experimental conditions.Then,based on the optimal internal genes,we explored the expression profile of four YUCCA genes in different organs and under different developmental stages and different abiotic stress conditions using qRT-PCR technology.Finally,according to the literature,the IiYUCCA6 gene was isolated and the gene function was preliminarily studied by constructing subcellular localization,prokaryotic expression and overexpression vectors.The obtained conclusions were as follows:(1)When comprehensively considering the result of geNorm and NormFinder software,IiPP2A-4,IieIF2 and IiRPL15 from 10 candidate reference genes were determined as the most stable reference gene for different developmental stages,different tissues and abiotic stress conditions,respectively.(2)The expression profile of four YUCCA genes(IiYUCCA2,IiYUCCA5,IiYUCCA6 and IiYUCCA8)was studied using qRT-PCR technology under different tissues and stages.According to the results,the expression level of IiYUCCA5 was highest in root,while the expression levels of IiYUCCA2,IiYUCCA6 and IiYUCCA8 were highest in senescent leaves and middle bolting stem;as to different developmental stages,the expression level of IiYUCCA6 was highest in flower period,while the gene expressions of IiYUCCA2,IiYUCCA5 and IiYUCCA8 were highest in seedling period.(3)The expression profile of four YUCCA genes(IiYUCCA2,IiYUCCA5,IiYUCCA6 and IiYUCCA8)was studied using qRT-PCR technology under different abiotic stresses.It showed that the drought stress could induce the expressions of IiYUCCA6 and IiYUCCA8 but inhibit the expressions of IiYUCCA2 and IiYUCCA5.The expression of IiYUCCA5 was reduced at initial stage but was promoted at later stage while IiYUCCA2,IiYUCCA6 and IiYUCCA8 were all promoted under salt stress.Low temperature could result in high expression of IiYUCCA8.However,low temperature stress showed a down-regulation at first and an up-regulation at last on the expression of IiYUCCA2,IiYUCCA5 and IiYUCCA6.Damage stress showed a consistent influence on all four YUCCA genes.The expressions were repressed at initial stage and then promoted at later stage.(4)IiYUCCA6 gene was isolated and analyzed.The bioinformatics analysis results indicated that the isoelectric point of IiYUCCA6 protein is 8.71.It belonged to the hydrophilic protein,which containing all the conservative motifs of FMOs monooxygenase and without the membrane structure domain or the signal peptide.The prediction results of subcellular localization showed that the protein might locate in cytoplasmic matrix.In addition,the amino acid sequence of Ii YUCCA6 was found to be more similar to that of other species of cruciferae by multiple sequence alignment and systematic evolutionary analysis,especially to that of Raphanus sativus.(5)In order to explore the subcellular localization of the Ii YUCCA6 protein,the corresponding plasmid was constructed and then disseminated into onion epidermal cells using agrobacterium tumefaciens-mediated transformation method.The observation results with fluorescence microscope showed that the proteins were mainly distributed in the cytoplasm.At the same time,a small amount was also distributed in the nucleus.(6)In order to obtain the optimal expression conditions of the fusion protein,the prokaryotic expression vector was constructed and transformed into E.coli.BL21.The results showed that the optimum condition of expression fusion protein were 0.5 mM IPTG,37 ? for 3 h.And the fusion protein was expressed in the form of inclusion body.(7)The overexpression vector was constructed and heterologously expressed in tobacco.The detection results in molecular level showed that the obtained lines were positive and the IAA content improved with increased plant heights and obvious apical dominance.Then,the positive line 3 and 9 were selected to detect the expression level of auxin response gene.The results indicated that the expression level of NtIAA8,NtLAA16,NtGH3.1 and NtGH3.6 increased significantly compared to the control group.Furthermore,the positive line 3 and 9 were exposed to the dark condition to detect the change of chlorophyll content,H2O2 content and the expression level of NtSAG12 gene.The experiment results showed that the decrease of chlorophyll content and the increase of H2O2 content were significantly reduced after 7 days in the dark.Besides,the expression level of NtSAG12 gene was significantly lower in the positive lines compared to the control group.All the above results indicated that the transgenic lines could delay the senescence of plants under dark conditions.