Isolation And Fuctional Analysis Of LrGLP1 Gene From Lilium Regale Wilson

Inevitable, plants are often suffered from a variety of biotic and abiotic stresses during their growth and development process. The innate immune response of plants has improved accordingly. Germin-like proteins (GLPs) are glucoproteins that ubiquitous and take parts in the development and defense-response of plants. In the present study, a novel GLP gene of Lilium regale Wilson was cloned based on an expressed sequence tags (EST) from the suppression subtractive hybridization (SSH) cDNA library of Fusarium oxysporum sp.lilii infected L. regale and designed as LrGLPl. A series of bioinformatics analyses of LrGLPl were carried out. The quantitative reverse transcription-PCR (qRT- PCR) was used for the analysis of the expression profile of LrGLPl in L. regale. Subsequently, recombinant protein of LrGLPl was obtained by prokaryotic expression and purificated by Ni-NAT clonm, and enzyme activities analyses of the LrGLP1 recombinant protein was carried out. Furthermore, the LrGLP1 was overexpressed in tobacco(Nicotiana tabacum L. cv Xanthi), and a series of studies were performed. The results of the present research were as follows:1. The full-length cDNA of LrGLP1 was cloned, and a series of bioinformatics analyses were carried out. The full-length cDNA of LrGLP1 was 908 bp in length and encoded a predicted polypeptide with 217 amino acid residues. The coding region of LrGLPl was disrupted by an intron of 103 bp. The results of bioinformatics analyses showed that LrGLP1 shared high identity with some known GLPs and belonged to the GLP subfamily 1. LrGLp1 contains the conserved domains of GLPs, and the 3-D structure of LrGLP1 was highly similar to that of the barley oxalate oxidase.2. The LrGLPl was tissue-specific expressed in L. regale with the higher transcrjptional level in the root. What’s more, the expression of LrGLPl in the root of L. regale was induced by the treatment of ethylene (ET), but down-regulated by the treatments of salicylic acid (SA), jasmonic acid (JA), and H2O2 at different degrees. The transcriptional level of LrGLP1 was dramatically up-regulated during the incompatible interaction between L. regale and F. oxysporum with the highest transcriptional level achieved at 24 h post-inoculation.3. The ORF of LrGLP1 was subcloned into pET-32(a) and expressed in Escherichia coli BL21 (DE3) with the induction of isopropyl-D-thiogalactopyranoside (IPTG). The recombinant protein was purified by the Ni-NTA column. Furtheremore, the enzyme activity analysis results showed that the LrGLP1 should be a bifunctional enzyme that has both superoxide dismutase (SOD) and oxalate oxidase (OXO) activities.4. The recombinant vector pCAMBIA2300S-LrGLP1 was constructed and transformed into tobacco, and the LrGLP1 transgenic tobacco lines were obtained. The results of qRT-PCR analysis showed that LrGLP1 was expressed in all the transgenic lines. What’s more, in the transgenic lines the expression levels of MnSOD, Cu/ZnSOD, PR-1b, and PR-1c were up-regulated, but the expression of CN was down-regulated. Under normal condition, the SOD and OXO activities in the leaves of transgenic tobacco lines were significantly higher than those of WT. What’s more, crude protein extracts of the leaves of LrGLP1 transgenic tobacco lines showed enhanced inhibition effects on the mycelium growth of F. oxysporum. Subsequently, both seeds and seedlings of LrGLP1 transgenic tobacco lines showed enhanced resistance to drought, NaCl, CdCl2, and F. oxysporum infection. All the stress treatments (drought, NaCl, CdCl2, and F. oxysporum) lead to the increased activities of SOD, peroxidase (POD), and OXO in the leaves of WT and transgenic tobacco plants. However, after treatments, the activities of all the 3 protective enzymes of the LrGLPl transgenic tobacco lines were significant higher than those of WT. Accordingly, MDA content and H2O2 accumulation in the LrGLP1 transgenic tobacco lines were significantly lower than those of WT.Overall, LrGLP1 was a novel GLP gene from L. regale that up-regulated by the ET treatment and F. oxysporum infection. And the LrGLP1 was a bifunctional enzyme that has both SOD and OXO activities. The ectopic overexpression of LrGLP1 in tobacco results in the changes of transcriptional levels of several resistance-related genes. Furthermore, the LrGLP1 transgenic tobacco lines showed enhanced ROS-scavenging capacity. As a result, the LrGLP1 transgenic tobacco lines had enhanced resistance to the drought, NaC1, CdCl2, and F. oxysporum infection. Therefore, the LrGLPl was a candidate gene that can be used for the genetic engineering technology to improve the resistance of plants.