| In the present study, a novel PRP gene of Juglans sigillata Dode was cloned based on an expressed sequence tag (EST) encoding the PRP from transcriptome sequencing data of Colletotrichum gloeosporioides infected J. sigillata by rapid amplification of cDNA ends (RACE) and designed as JsPRP1. A series of bioinformatics analyses of JsPRP1 were carried out. The quantitative reverse transcription-PCR (qRT-PCR) was used for the analysis of the expression profile of JsPRP1 in J. sigillata. Subsequently, recombinant protein of JsPRP1 was obtained by prokaryotic expression and purificated by Ni-NAT clonm, and antifungal activities analyses of the JsPRP1 recombinant protein was carried out. Meanwhile, the pBIN m-gfp5-ER vector of JsPRP1 gene was constructed, and then the recombinant vector were transformed into Onion(Allium cepa L.) epidermal cells by Agrobacterium tumefaciens-med’iated, and using green fluorescent protein signal to determine the distribution of target proteins in cells by the laser scanning confocal microscope. Furthermore, the JsPRP1 was overexpressed in tobacco (Nicotiana tabacum L. cv Xanthi), and many studies were performed. The results of the present research were as follows:1. The full-length cDNA of JsPRP1 was cloned, and a series of bioinformatics analyses were carried out. The full-length cDNA of JsPRP1 was 815 bp in length and encoded a predicted polypeptide with 135 amino acid residues. The results of bioinformatics analyses showed that JsPRP1 belongs to the PRP family. The 3-D structure showed that JsPRP1 consisited of four a-helix and loop, and cross-helix formed a hole.2. The qRT-PCR analysis showed that JsPRP1 was induced after inoculation with C. gloeosporioides, and the highest transcription level was achieved at 48 h post inoculation (hpi). The transcriptional level of JsPRP1 was quickly induced by the treatment of jasmonic acid (JA) at 6 hpi and 24 hpi, and the level was dramatically up-regulated by the treatments of salicylic acid (SA), ethylene (ET), and H2O2 with the highest transcriptional level achieved at 48 hpi.3. The ORF of JsPRP1 was subcloned into pET-32(a) and expressed in Escherichia coli BL21 (DE3) with the induction of isopropyl-β-D-thiogalactopyranoside (IPTG). The recombinant protein was purified by the Ni-NTA column. Furthermore, the activity analysis of the JsPRP1 fusion protein results showed different levels of resistance against Gibberella moniliformis, C. gloeosporioides, Botrosphaeria dothidea, and Fusarium solani.4. The pBIN m-gfp5-ER vector of JsPRP1 gene was constructed, and then was transformed into Onion(Allium cepa L.) epidermal cells by Agrobacterium tumefaciens-mediated, and determined the distribution of target proteins in cells by the laser scanning confocal microscope. Subcellular localizatiom assays showed that the JsPRP1 protein appeared in the cell wall.5. The pCAMBIA2300S-JsPRP7 was constructed for stable transformed of tobacco plants. The JsPRP1 transgenic plants were obtained and T2 generation transgenic plants were obtained. The results of qRT-PCR analysis showed that JsPRP1 was expressed in all the transgenic lines steadily. Compared with wild type (WT), the JsPRP1 T2 generation of transgenic tobacco plants improved tolerance to drought, CdCl2, and C. gloeosporioides infection. Accordingly, under the drought and CdCl2 strsses, the H2O2 accumulation in the JsPRP1 transgenic tobacco lines were significantly lower than those of WT.Overall, JsPRP1 was a novel PRP gene from J. sigillata that up-regulated by the signal molecules treatment and C. gloeosporioides infection. And the JsPRP1 was a antifungal activity enzyme. Subcellular localizatiom assays showed that the JsPRP1 protein appeared in the cell wall. The ectopic overexpression of JsPRP1 in tobacco significantly increased the resistance to drought, CdCl2, and C. gloeosporioides infection. Therefore, the JsPRP1 is a candidate gene that can be used for the genetic engineering technology to improve the resistance of plants. |
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