| Postharvest disease of fruit caused by the fungal pathogen infection, results in significant economic losses. Thus, the control of postharvest diseases has an important role to reduce the losses. Biological control with antagonistic yeasts has been considered an effective method to control the postharvestdiseases of fruit because they are perceived as environmentallysafer and more acceptable to the general public. Antagonistic yeasts can stimulate resistance responses of host plant, the induction of resistance of fruit to reduce postharvest diseases is an important strategy. This is attractive because it uses the defense mechanisms of the plant itself and has abroad-spectrum antibacterial property. In the previous work of our group, we used Affymetrix Tomato Genechip microarrays to evaluate changes in gene expression in response to Cryptococcus laurentii in cherry tomato fruit. LePR5gene was significantly up-regulated. Thus, LePR5protein induced by C. laurentii was the major anti-protein involved in the regulation of resistance responses of the fruit. This study explored the LePR5gene expression patterns of fruit induced by C. laurentii and Alternaria alternata, verified the inhibitory function LePR5protein which expressed by prokaryotic protein expression system, confiremed the relevance of the protein antibacterial function and the biocontrol effect of C. laurentii. The LePR5promoter region gene sequence was cloned and its promoter functional activity was verified. The LePR5promoter region gene sequence was analysed through software, screened the cis-acting elements in the promoter region of LePR5gene and transcription factors with the gene microarray result. The expression regulatory mechanism of LePR5gene which was induced by C. laurentii was studied. The feasibility study of combined application of C. laurentii and MeJA to enhance the disease resistance was also implemented.The main results are as follows:(1) The expression pattern of LePR5gene transcription level was analysed using qRT-PCR which was induced by C. laurentii, and the expression pattern was compared with another one which was induced by the pathogen, A. alternata. The results showed that the relative transcript level of LePR5was increased gradually in response to C. laurentii and A. alternata. After24h, the relative level of LePR5transcripts was significantly up-regulated greater than2.0fold (P<0.05) for C. laurentii and A. alternata compared to non-infection fruit, LePR5gene as a resistance-related gene was involved in resistance response of fruit. The3D structure of LePR5protein was predicted and constructed using bioinformatics software, then the3D structure and amino acid sequence of LePR5protein was analysed, the results showed that high similarities were found in the amino acid sequences between LePR5protein and other PR5antifungal proteins, the conserved functional elements are present in the LePR5protein. These results indicated that LePR5protein have biological function.(2) The LePR5recombinant protein was obtained by E.coli expression system, and LePR5recombinant protein was verified by Western-bloting. The anti-fungal activity of purified LePR5recombinant protein was studied in vitro and in vivo.10,20and40μg LePR5recombinant protein treatment reduced the disease incidence approximately18%,31%and49%comparing with the control (100%) separately on the5th day after inoculation. It indicated that the efficiency of LePR5recombinant protein in controlling the black rot is in a dose-dependent manner.(3) The promoter sequence fragment of LePR5gene was cloned and the sequence was constructed into the expression vector containing GFP/GUS reporter gene, then the expression plasmid was transformed into protoplast and observed the expression of green fluorescent protein (GFP) in the transient expression system. Meanwhile, the cherry tomato transgene plants which transformed with PLePR5:GFP/GUS expression plasmid were constructed, and staining showed GUS expression. These results showed that the cloned sequence has promoter activity.(4) The cloned promoter fragment sequence was analysed using the PLACE database, the results showed that there are5cis-regulatory elements and transcription factor binding sites, mainly for GCC box and W box, which were related with PRs. Combined with cherry tomato microarray results, we screened GCC box as the cis-regulatory elements and Pti5as the transcription factor for regulating the expression of the LePR5gene.(5) The expression pattern of the Pti5gene transcription level was analysed using qRT-PCR which was induced by C. laurentii and A. alternata. The results showed that the relative transcript level of Pti5was increased gradually in response to C. laurentii and A. alternata. The Pti5recombinant protein was obtained by E.coli expression system, and Pti5recombinant protein was verified by Western-bloting. The promoter fragment containing GCC box of LePR5gene was cloned and prepared to be biotin-labeled probe, meanwhile, synthesized GCC box repeated sequence probe and mutated GCC box repeated sequence probe. EMSA results showed that Pti5transcription factor proteins could bind to the LePR5gene promoter fragment containing GCC box and also the GCC box repeated sequence, but can not bind to the mutated GCC box repeated sequence. These results suggested that C. laurentii induced the expression of Pti5gene to activate the expression of LePR5gene in cherry tomato fruit. We also analyzed the expression of Pti5gene and LePR5gene under the condition of hormone (MeJA) induced separately and the combined application of C. laurentii and MeJA, the results showed that Pti5gene did not respond to MeJA. The expression of LePR5gene was upregulated under the condition of MeJA treated separately and combined treatment of C. laurentii and MeJA, the combined treatment has synergistic effects in the expression of LePR5gene.(6) Citrus as test materials, the feasibility and mechanism of the combined treatment of biocontrol yeasts and MeJA was studied. The results showed that100μmol/L MeJA inhibited disease incidence and lesion diameter of mold decay compared with the control (P<0.05). The preventive application of C. laurentii at1×108cells/mL combined with100μmol/L MeJA reduced green mold incidence compared to the controland the other treatment groups when tested in wounded citrus fruit inoculated with Penicillium digitatum. MeJA at a certain range of concentrations has no direct impact on the growth of P. digitatum, the population of C. laurentii was greatly increased by combination with MeJA in vitro and in vivo. MeJA and C. laurentii induced higher activity of polyphenol oxidase, peroxidase and catalasethan control. Moreover, treatment with MeJA and C. laurentii induced a rise in the mRNA expression levelof PR5, which was stronger than in the single-treatment groups and the control. |
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