| Plants often suffer from various abiotic and biotic stresses during growth and development,such as drought,low temperature,pathogens,and so on.Therefore,plants have evolved a battery of defense mechanisms to combat these stresses.For example,the activation and accumulation of pathogenesis-related protein 10(PR 10)plays important roles in plant defense response.In this study,nine expression sequence tags(ESTs)encoding PR10s were isolated from a suppression subtractive hybridization(SSH)cDNA library of Lillum regale inoculated with Fusarium oxysporum f.sp.lilii.By using rhe rapid amplification the cDNA end(RACE),the full-length cDNA sequences of PR10s in L.regale Wilson were cloned.To understand the LrPRIO gene family and the characteristics of LrPR10s,the bioinformatics analyses were performed.The expression properties of LrPR10s were detected by quantitative real-time reverse transcriptase PCR(QRT-PCR)analyses.The prokaryotic expression vector of LrPR10-5 gene was constructed.The recombinant LrPR10-5 protein was purified,and the RNase activity of recombinant protein was detected in vitro.A promoter fragment of LrPR10-5 was cloned from L.regale using genomic walking technique and,the promoter cis-elements were predicted.The 5'delection fragments of LrPR10-5 promoter was fused to ?-glucuronidase(GUS)gene to constructe plant expression vectors,and then were transformed into tobacco(Nicotiana tabacum L.cv Xanthi)by Agrobacterium tumefaciens-mediated method,respectively.To understand the function of the promoter,the transgenic tobaccos were treated by plant hormones,abiotic and biotic stresses and the promoter activity were detected by quantitative fluorometric assays.Furthermore,the plant overexpression vector of LrPR10-5 was constructed and transformed into tobacco.The T1 generation of transgenic tobacco was used to study the resistance against fungal pathogens and the activity of RNase.The main results are indicated as follows:1.Nine full-length cDNA sequences of PR 10 genes were isolated by RACE.The bioinformatics analysis demonstrated that the full-length cDNAs of L.regale PRIOs ranged from 696 bp to 956 bp,and the predicted amino acid residues of LrPR10s were 156 or 157.Multiple alignments showed that most of the L.regale PR10s contained a 'P-loop' motif and three conserved amino acid residues.Phylogenetic analysis displayed that the nine LrPR10s with other PR10s from monocotyledon species were clustered into one branch,and nine LrPR10s were formed two different sub-groups.2.QRT-PCR analysis revealed that the highest expression levels of LrPRIOs were detected in healthy L.regale roots,while relative weakly in the leaves and stems.The jasmonic acid,salicylic acid,ethylene and H2O2 inhibited the expression of LrPR10-3,LrPR10-4,LrPR10-8 and LrPR10-9,while induced the expression of the other LrPR10s.The expression profiles of the nine novel L.regale PR10s in the resistant L.regale and susceptible Lilium Oriental Hybrid 'Siberia' were not identical after inoculation with F.oxysporum.The expression levels of LrPR10-2,LrPR10-4,LrPR10-5,LrPR10-6,LrPR10-7 and LrPR10-9 were induced significantly by F.oxysporum in L.regale,while others were induced in 'Siberia'.3.The pET-32a-LrPR10-5 was constructed and then transformed into E.coli BL21(DE3).The LrPR10-5 recombinant protein(about 27 kDa)was isolated in solubility under 0.1 mM IPTG at 30 ? inducing for 4 h.The fusion protein was purified by Ni-NTA agarouse.The RNase activity assay showed that the purified LrPR10-5 protein possessed RNase activity in vitro.4.A 1 533 bp promoter fragment of LrPR10-5 was cloned from L.regale Wilson using genomic walking technique.The bioinformatics analysis indicated that the fragment contained a series of elements related to stresses.To determine the expression pattern of the promoter,the promoter was deleted from its 5' end to form promoter fragments,and the above fragments were fused to GUS gene.The fused genes were transformed into N.tabacum using A.tumefaciens-mediated method to obtain stable expression transgenic plants.Based on the cis-elements,the transgenic plants were treated by biotic,abiotic stresses and several plant hormones.GUS quantitative fluorometric assays indicated that the three fragments of the promoter had promoter activities and the GUS activities in the roots of transgenic plants were increased with the increasing length of promoters.The LrPR10-5 promoter responded to several abiotic and biotic stresses,as well as a few plant hormones.5.pCAMBIA2300S-LrPR10-5 was constructed for stable transformation of tobacco plants.Then the LrPR10-5 transgenic plants were generated and T1 generation transgenic plants were obtained.The QRT-PCR analysis showed that the LrPR10-5 gene was steadily expressed in the T1 transgenic tobacco lines.Antifungal activity of the crude protein extracts of the LrPR10-5 transgenic tobacco lines displayed different levels of resistance against Botrosphaeria dothidea,F.oxysporum and Sclerotinia sclerotiorum.Otherwise,the five LrPR10-5 transgenic tobacco lines demonstrated enhanced resistance of against F.oxysporum inoculation assay in vivo.The RNase activity assay established that the RNase activity of LrPR10-5 transgenic tobacco lines was up-regulated by F.oxysporum inoculation.In summary,the nine L.regale PR10 genes were induced by several signaling molecules and F.oxysporum.The LrPR10-5 expression was regulated by JA.ET and H2O2,and inducted by F.oxysporum.The recombinant LrPR10-5 protein possessed RNase activity in vitro.The promoter activity was enhanced under several plant hormones,biotic and abiotic stresses.The overexpression of LrPR10-5 in tobacco enhanced the resistance to F.oxysporum significantly The RNase activities of transgenic tobacco lines were up-regulated by F.oxysporum infection.Therefore,the cloning and function analysis of L.regale PR 10 gene family made a contribution to study the molecular mechanism of resistance in L.regale,and provided several available resistance genes for plant gene engineering. |
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