Cloning And Functional Analysis Of Serine Carboxypetidases Gene From Zea Mays L.

Plants were often invased by some pathogenic microorganisms through the long-term evolution, and they co-exist for a long time, interact each other and even the co-evolution in ecosystem. Plants gradually form a series of plant defense mechanisms to protect themselves. Plant disease resistance is a very complex process and involved in a variety of regulation and defense responses, including the identification of pathogens, signal transduction, gene regulation and expression of disease resistance and so on. Serine carboxypeptidase gene (SCP) and serine carboxypeptidase like (SCPL) gene play an important role in plant wound responses and the synthesis of brassinosteroids. In this study, silico cloning, reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were employed to clone the serine carboxypeptidase gene from Zea may L.; The expression pattern of target gene was analyzed with the semi-quantitative PCR and real-time PCR; The expressed protein of target gene was purified and analyzed the antifungal properties; We construced the over-expression vector for the further research of target gene's function. The main results are as follows:(1) Silico cloning, reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were applied to clone the serine carboxypeptidase gene using cDNA of the high resistant material R15 induced by Rhizoctonia solani. The cDNA full length of serine carboxypeptidase gene is 1874 bp (Genbank accession number:JF682634) containing a 999 bp complete open reading frame, which is from 154 bp to 1152 bp, a 153 bp 5'untranslated region, a 722 bp 3' untranslated region. ZmSCP has continuous poly A and a typical polyadenylation signal. Bioinformatical analysis showed ZmSCP codes 333 amino acid, the size of expected encoded proteins is 36.505 KD, the isoelectric point is 4.75. The homology analysis indicated that the deduced amino acid from zea mays L. shares different homology with other plants whose homology percentage of the range are from 42% to 81%. Phylogenetic analysis revealed ZmSCP has closer kinship with Oryza satiua and sorghum, they belong to the same evolutionary branch. It was named ZmSCP.(2) The type of amino acids, protein modification sites, signal peptide region, sub-cellular localization and conserved domains of ZmSCP gene were predicted and analyzed by bioinformatics in this study. The results are as follows:There are four types of amino acids in ZmSCP protein, and the proportion of hydrophobic amino acids is highest among the four amino acids; ZmSCP protein has four protein modified ways which include an N-glycosylation site, two protein kinase C phosphorylation sites,2 casein kinaseⅡphosphorylation sites, eight N-myristoylation sites and no signal peptide region in protein sequences; Sub-cellular localization demonstrated that the ZmSCP protein might mainly express in the cytoplasm. In addition, ZmSCP protein has S10 conserved domain and belongs to S10 superfamily.(3) Maize genomic DNA from R15 was extracted and then digested by four kinds of restriction enzymes (EcoRⅠ, NotⅠ, SpeⅠand BglⅡ). Southern blot suggested that four lanes appear several hybridization bands. ZmSCP gene may belong to multiple copies.(4) The maize inbred line R15 was used as material, the target genes mRNA expression of ZmSCP genes were analyzed by semi-quantitative and quantitative methods under different stress conditions. It is showed that the ZmSCP gene are basically up-regulated expression after treatment. Of which, ZmSCP gene expression showed two-step trend induced by Rhizoctonia solani, It reached the first peak after inoculation in 24 h then decreased, and the second peak in 60 h, and it appeared significant differences compared with the non-inoculated. In addition, the expression of ZmSCP gene increased under the ABA, JA, cold and salt stress and appeared expression peak in 48h.(5) Maize R15 inbred lines seedlings which induced by Rhizoctonia solani AG1-IA were used as materials, open reading frame of ZmSCP gene was amplificated by RT-PCR, cloned into pMD18-T vector and subcloned into pET32a (+) expression vector to obtain the prokaryotic expression vector pET32a(+)-ZmSCP. The recombinant expressed plasmid pET32a(+)-ZmSCP was transformed into E. coli BL21 after identifying correctly. The expression system was optimized at different temperatures (28℃,37℃), with different concentrations (0,0.2,0.4,0.6,0.8,1.0 mmol/L) of IPTG, for different induction time (0,2,4,6,8,10 h). The optimum expression condition is after 6 h treatment of 0.6mmol/L IPTG at 37℃. The expression products migrated at the size of 58 kD via SDS-PAGE and western blot. In conclusion, protein expression have successfully proved.(6) ZmSCP protein was purificated by Ni-NTA resin, using PBS solution as control, three different protein concentrations were set and co-cultured with Rhizoctonia solani. The growth of mycelium was observed and taken a photograph after 24h, meanwhile, we measured the size of inhibition zone and observed the growth of mycelium under the microscope. It is showed that fusion protein inhibited the growth of Rhizoctonia solani when the proein is 20μg and inhibition became obvious as the increase of ZmSCP protein. The size of inhibition zone were positively correlated with the concentration of fusion protein. What's more, however the growth of mycelium and hyphal density were decreased with the increase of protein concentration under microscope.(7) In order to analyze the function of ZmSCP gene, we constructed the over-expression vector pCAMBIA 1301-ZmSCP (promoter is 35S) and transform into tobacco. In this study, twenty-one kana-resistant lines were obtained and genomic PCR, semi-quantitative RT-PCR and SDS-PAGE revealed seven lines were positive transgenic tobacco plants which named line one-line seven. In addition, the ratio of resistant seedlings and un-resistant seedlings on kana-resistant MS medium was 3:1 which suggested ZmSCP can inherit and express stably in offspring.(8) It is showed that transgenic tobacco T1 plants improved resistance to Rhizoctonia solani, tobacco pathogen, tobacco bacteria disease and the expression of defense-related genes NtPRl, NtPR2, NtPR3, NtPR5 and NtPAL increased in the transgenic plants. In addition, the transgenic plants shown some resistance to abiotic stress (such as drought, high salt, oxidation). It is indicated that the ZmSCP gene showed broad spectrum resistance and plant disease resistance involved in a variety of signal transduction pathway and the joint of many proteins.