Cloning, Characterization Of A Tall Fescue Class Ⅰ Chitinase Gene (FaChit1) And Functional Analysis Of Its Promoter; Electrochemical Detection Of The Transgenic Plants With A Novel DNA Biosensor

Turf grasses have wide applications in lawn construction,with the purpose to beautify living conditions and enrich diverse demands of people.However,most Turf grass species bear poor adverse-environment-tolerance and easy to suffer from plant diseases and insect pests,especially from pathogenic fungus.Nowadays,it is an urgent task to improve the abilities of diseaseresistance, drough-resistance,salt and barrenness-tolerance in cultivation of high-quality turf grasses.It seems to be a good way to transfer plant chitinase gene into host plants need to be improved by transgenic technology.It is well known that plant chitinase,especial class I chitinase, can effectively degrade chitin,a main component of cell wall in fungi and insect,to inhibit the growth of pathogens.And some chitinases possess other tolerance characteristics,which can greatly enhance the adaptability of plants.However,this kind of available gene resource is not enough for ulitization,also lack of revelant safty evaluation.It was found that Festuca arundinacea,which belongs to the family Gramineae,possesses the characteristics of strong disease-resistance,drought-resistance,barrenness-tolerance and high adaptability.Therefore,it was proposed that if a class I chitinase gene along with its promoter from Festuca arundinacea could be isolated and well characterized functionally,and was followed by transferring them together into some turf grasses need to be improved genetically,then,the resistance of turf grasses to pathogens and environmental stresses would be improved to a great extent.Besides,as transgene was expressed under the controll of stress-inducible promoter,not in constitutive manner,renderring transgenic plant to be highly-adaptable to adverse environment.Based on this proposal,we sought to isolated and characterize a class I chitinase gene and its promoter from Festuca arundinacea in order to look into molecular events associated with resistance and provide available resistance-related gene for plant genetic engineering.At first,a conservative fragment of 849 bp was amplified from Festuca arundinacea cDNAs using degenerate primers with homology to plant chitinases.A full-length cDNA of Festuca arundinacea class I chitinase gene was obtained using 5' RACE and 3' RACE,which named as FaChitl and registered in GenBank(accession number EU837265).The full-length open reading frame contained 951 base pairs and encoded a deduced class I chitinase containing 317 amino acids with a calculated molecular mass of 33.24 kDa and a pI of 7.91,which was consistent with the properties of most previously published plant class I chitinases.BLAST searches indicated that the putative FaChitl has three conserved amino acid motifs,a chitin binding domain(CBD), a proline and glycine-rich hinge region(HR),and a catalytic domain(CD).Consistent with other plant class I chitinases,no deletion was observed in the CD of FaChitl.Interestingly,no carboxyl-terminal extension(CTE) specific to most class I chitinases was found in the putative FaChitl.To investigate the structure of FaChitl,genomic DNA corresponding to its coding region was cloned and no intron was found.To investigate its putative promoter,a 5'-flanking region containing 935 bp sequences upstream of the FaChitl start codon was isolated from Festuca arundinacea genomic DNA using Genome walking method.Sequence analysis of this FaChitl promoter fragment using the PLACE program identified typical TATA and CAAT boxes 120 bp and 259 bp upstream of the start codon,respectively.Besides,Several potential cis-acting elements associated with plant hormone and stress-related responses were located within the FaChitl promoter,such as W-box,ABRE,MYB transcription factor binding sites and MYC transcription factor binding sites,indicating that this promoter might be multiple stress-inducible. Southern blotting revealed that there were two copies of FaChitl in Tall feseue genome.To determine the expression profile of FaChitl under different stress conditions.RNA gel analyses using a FaChitl coding region probe were done.Northern blotting revealed that FaChitl-mRNA was barely detectable in leaves and roots of untreated plants.By contrast,high amounts of FaChitl-mRNA were detected in leaves and roots treated with fungal elicitors. FaChitl expression was also induced in both tissues by ethylene and mannitol,and to a lesser degree,by mechanical wounding in roots,but not in leaves.To further investigate the regulation of FaChitl,activation of different promoter fragments by fungal elicitors,dehydration,mechanical wounding,and ethylene were analyzed in transgenic tobacco using transcriptional fusions of FaChitl 5' upstream sequences to the GUS reporter gene (promoter-GUS expression vectors were designated as pFaChitlP-Ⅰ,pFaChitlP-Ⅱand pFaChitlP-Ⅲ,respectively).Compared to mock treated controls,the 935 bp and 651 bp DNA sequences upstream of the FaChitl start codon conferred 6.5- and 5.1-fold inductions, respectively,of GUS activity in response to a 24-hr treatment with fungal elicitors.However,no induction of GUS activity could be detected in transgenic tobacco leaves containing the 233 bp upstream sequence.Under dehydration stress,GUS activities were 5.4-,4.75-,and 2.25-fold induced in leaves of tobacco harboring the 935 bp,651 bp,and 233 bp upstream sequences, respectively,compared to mock treated controls;Spraying of plants with ethylene increased GUS activity 5.0-,2.5-fold in leaves of transgenic tobacco harboring the 935 bp,651 bp sequences, respectively,but no GUS activity could be detected in transgenic tobacco leaves containing the 233 bp upstream sequence,compared to mock treated controls.Compared to mock treatment, GUS activity could also be induced moderately 2.4- and 2.1-fold,respectively,in response to mechanical wounding in leaves containing the 935 bp and 651 bp sequences,but not in plants with the 233 bp upstream sequence.Anlysis of promoter deletion indicated that the fragment between 935 bp and 233 bp upstream of the FaChitl start codon was sufficient to direct the gene expression in response to fungal elicitors,ethylene,dehydration,or mechanical wounding due to both known and presently uncharacterized cis-acting elements.Besides,a novel DNA electrochemical biosensor was developed and characterized for the detection of NPT-Ⅱ,a selective marker widely used for transformation of many plant species mediated by Agrobacterium tumefaciens.Our biosensor possesses a feature of universal utility in the identification of transgenic plants.Experiment results indicating,specificity of biosensor was significantly improved by using thiol-modified capture and biotinylated signaling probes complementary to target regions flanked by PCR primers.The detection sensitivity was greatly enhanced by the dendritic enzymatic magnification of the hybridization signal.The limit of detection(LOD) was 0.2×10^-9 mM.In summary,the biosensor designed in this work could provide a platform for the establishment of a reliable,accurate,sensitive,and efficient detection system for emerging transgenic plants.