| Spraying salicylic acid can greatly induce the expression of stresses-related genes of plant, which would enhance the plant’s resistance to adversity stresses such as biotic stress (disease and insect Pests, e.t.c) and abiotic stress (meteorological disasters and soil stress, e.t.c), having the advantages of green, environment protection and efficient and will become a new approach to control plant disease and insect pests and improve the flexibility of plant to environmental stresses. Isolation of the pathogen- and stresses-related genes that can be transcriptionally induced by exogenouse salicylic acid has important theoretical significance and practical value to genetic improvement of plant disease and insect pests control and improvement of adaptability of adversity stress. "Nai"(Prunus salicina) belongs to the prunus family, Rosaceae, and are believed to be originated from Fujian province of China. "Nai" is one of the famous, special and excellent stone fruits trees, which has important economic value. In this study, the leaves of the one year grafted seedlings of "Nai" were sprayed with exogenous salicylic acid of different concentrations. The salicylic acid treated leaves were harvested for total RNA extraction at different time-ponts after treatment; The dilution pool PCR method was used to screen leaves with a normalized full-length cDNA library and to obtain the full-length cDNA of the homologous genes of NPR1 and WRKY which are related with the salicylic acid with induced resistance. The different transcript expression levels of these genes in response to different concentration os salicylic acid were detected via using the real-time qPCR and to confirm the optimum concentration of salicylic acid to induce the expression of resistance-relative genes. The confirmed optimum salicylic acid was used to spray "Nai" seedlings.30.0 hours and 0.0 hours after treatment, the "Nai" leaves were harvested to construct the cDNA library of DSN mediated-Suppression Subtractive Hybridization. Reverse Northern-Blotting was used to screen the Suppression Subtractive Hybridization cDNA library and then to obtain the positive clones. The positive clones were sequenced and analyzed by bioinformatics. Real-time Fluorescence Quantitative PCR system was usde to verify the positive clones of differentially expressed. The main results are as follows:1. The isolation of cDNA full-length of homology genes NPR1 and WRKY of the "Nai" Leaves:The dilution pool PCR method was used to screen leaves with a full- length normalized cDNA library and four NPR1 of the full-length and twelve WRKY homology genes were obtained. The sequences of these acquired genes were analyzed by bioinformatics method. Real-time Fluorescence Quantitative PCR technique was used to compare the dynamic changes of expression amount in spraying different concentrations of salicylic acid (SA) and at different response time.(1) Four cDNA full-length of NPR1 homology genes were acqured from the "Nai" leaves in our study, which are named as PsNPR1, PsNPR3.1, PsNPR3.2 and PsNPR3.3, respectively. Their lengths are 2442 bp、1827 bp、1920 bp and 2615 bp, respectively. Their ORF lengths are 1794 bp、1777 bp、1773 bp ' 1788 bp, respecitively and encoded as 596,591,589 and 594 amino acids, respectively. Their 5’-UTR lengths are 114 bp、32 bp、144 bp and 573 bp and their 3’-UTR lengths are 114 bp、32 bp、144 bp and 254 bp, respectively. The homology of PsNPR1 and Pyrus serotina NPR1 is 84%. The homology of PsNPR3.1 and Malus domestica NPR3 is 82%. The homology of PsNPR3.2 and prunus persica NPR3 is 95%, the PsNPR3.3 and prunus mume NPR3 is 98%.The amino acid sequence analysis results showed that four NPR1 homologous genes encoding region contained the BTB/POZ and ANK conservation domain and the nuclear localization signal (NLS). The analysis results of phylogenetic tree showed that the PsNPR1 and Arabidopsis thaliana of AtNPR1 and AtNPR2 gather into one cluster. PsNPR3.1, PsNPR3.2 and PsNPR3.3 and Arabidopsis AtNPR3 and AtNPR4 together gather into another cluster.(2) The reaserch have obtained twelve cDNA full-lengths of WRKY homology genes from the "Nai" leaves, which are named PsWRKY7, PsWRKY14, PsWRKY20, PsWRKY21,PsWRKY22, PsWRKY29, PsWRKY31, PsWRKY32, PsWRKY33, PsWRKY40,PsWRKY46 and PsWRKY75, respectively. Their length are 1517bp、1798 bp、2098bp、1177bp、1242bp、1396bp、2135bp、1760bp、2113 bp、1047bp、 1258 bp and 700 bp, respectively. Their ORF lengths are respective 11095 bp、1572 bp、1764 bp、1068 bp、1071 bp、1002 bp、1944 bp、1602 bp、1770 bp、963 bp、 1077 bp and 672 bp. They are encoded as 364,523,587,355,357,333,647,533,589, 320,358,223 amino acids, respectively. After blast with Arabidopsis genome database, it was found that they are first to match respectively to AtWRKY7, AtWRKY14, AtWRKY20, AtWRKY21,AtWRKY22, AtWRKY29, AtWRKY31, AtWRKY32, AtWRKY33, AtWRKY40, AtWRKY46 and AtWRKY75. They all belong to the WRKY transcription factor. The amino acid sequence analysis results showed that these genes were found to contain WRKY conservation domain. The analysis results of phylogenetic trees show that PsWRKY20, PsWRKY32 and PsWRKY33 belongs to Group I and PsWRKY7, PsWRKY14, PsWRKY21, PsWRKY22, PsWRKY29, PsWRKY31, PsWRKY40 and PsWRKY75 belongs to Group II, and PsWRKY46 belongs to Group III.2. The determination of the optimal concentration and response time to spray the salicylic acid:Real-time Fluorescence Quantitative PCR technology was used to compare the difference of expression of PsNPR1 in different concentrations of salicylic acid (0,0.005,0.05,0.5,1 and 2 mM) 24 hours after spraying and 1.0 mM was determined to be the optimum concentration of salicylic acid treatment. After comparing the difference of expression of PsNPR1 and PsWRKY at spraying "Nai" leaves with 1.0 mM salicylic acid 0.0,1.0,3.0,6.0,12.0,24.0,30.0,36.0,48.0 and 72.0 h post treatment to determine the optimal response time of salicylic acid. The results show that PsNPR1 and PsWRKY both exhibited the highest expression levels 30.0 hours after spraying with salicylic acid 1.0 mM, except PsWRKY14. So the most suitable concentration of salicylic acid spraying was 1.0 mM and the best response time after spraying was 30.0 hours.3. The isolation of differential genes with associated with the abduction of exogenous salicylic acid induced in the resistance of "Nai" leaves:"Nai" leaves spraying 1.0 mM salicylic acid after 30.0 hours and 0.0 hours was used as the material to construct DSN mediated-Suppression Subtractive Hybridization. The positive clones were screened by reverse Northern-blotting technology. According to the results of sequencing of the positive clones, the primers were designed. Comparing the differential expression of these positive clones of 1.0 mm salicylic acid after 30.0 hours and 0.0 hours by Real-time fluorescence quantitative PCR method to confirm the positive clone of the differential genes in the induction of the exogenous salicylic acid. Analyzing the results of the sequencing of positive clones by bioinformatics information method will be benefit to predict the function of these genes. The test results show that the optimal concentration of DSN was 1/2 U and the optimal cycle number of amplified by LA-PCR of treated products of DSN was 22. The difference of amplification products of cDNA concentrated in the range of 0.5-3.0 kb and the dispersion does not appear bright band, which indicates that DSN treatment can effectively remove the cDNA of the same genes, which can achieve an effect of subtractive and normalized. The result of electroporation indicates that the initial titer of the Suppression Subtractive Hybridization cDNA library was 1.7×106 pfu/mL. Selected randomly 16 clones to make verification of colony PCR, whose results show that insert size is in the range of 1 kb-3 kb,each colony has bright special band and the average insert size is 1.7 kb of this library.190 clones were chosen randomly to make reverse Northern-blotting and obtained 38 positive clones. The positive rate was 20%. The results of sequence of positive clone showed that there are two repeated in the 38 sequences. The redundancy rate is 2.6%; the results of positive clone by Real-time Fluorescence Quantitative PCR showed that in 37 positive clones, except Ps20 clone and Ps29 clone, there are significant differences in their relative expression levels in the other positive clones on the leaves with salicylic acid spraying after 30.0 h and 0.0 h leaves. The result is consistent with the reverse Northern-blotting screening.37 obtained genes was ananlyzed and functional annotated using BGI, WEIGO, BLAST and the results showed that in the 37 genes, there are 11 genes involved in cell composition which accounted for 29.7%,there are 6 genes molecular function which accounted for 16.2%, there are 19 genes involved in biological processes (51.4%) and 1 unknown gene accounted for 0.7%; in the 37 genes, there are 8 genes involved in biotic stress response,10 genes involved in abiotic stress response,18 genes involved in the response to endogenous stimulation and drug stimulation; in the 37 genes, there are 8 transcription factor genes involved in biotic stress and abiotic stress response: GRAs family, ethylene response protein gene,TGA, WRKY and BHLH. |
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