Cloning And Preliminary Functional Analysis Of Gmtlpin Soybean

Soybean [Glycine max (L.) Merr.] is one of the most important sources of plant protein and edible oil world-widely, which occupies an important position in the food and agricultural industry. Soybean mosaic virus (SMV) is one of the most prevalent and important plant viral pathogens imperiling legume crops worldwide. Infection by SMV may result in severe yield losses and quality degradation. To the present, croppers reduce the impact of SMV on soybean yield mainly through appropriate planting period, the selection of resistant varieties, anti-aphid treatment and other measures. Thaumatin-like protein is an important member of the group5pathogenesis-related proteins, which shows direct antifungal action against a broad range of fungal pathogens.We cloned two copies of thaumatin-like protein in soybean, which named GmTLPl and GmTLP2. In this study, the expression and function of the thaumatin-like protein gene GmTLPl, isolated from soybean (Glycine max L.) cultivar KeFeng, were characterized. All results are as follows.1. The expression of GmTLPl gene was found to be significantly up-regulated by SMV infection at4h,8h,24h, and48h after treatment using real time fluorescence quantitative PCR. And the transcriptional profile of GmTLPl gene response to SMV infection was a bimodal pattern.2. The expression pattern of GmTLP1in different organs was examined using semi-quantitative RT-PCR analysis and transcripts of GmTLPl gene could be detected in all organs, but with low-level expression in root.3. The green fluorescent protein expression vector, GmTLP1-GFP-pMDCS3, was constructed and transformed into the epidermal cells of onion via Agrobacterium-mediated method. In the transformation of the empty vector pMDC83-GFP, green fluorescence was observed throughout the onion epidermal cell. However, in the transformation of recombinant plasmid pMDC83-GmTLP1-GFP, green fluorescence was observed only in the cell membrane during plasmolysis process by a certain concentration of NaCl. The results of instantaneous expression showed that GmTLP1proteins were localized in cell membrane.4. High-level expression of GmTLPl was observed in transgenic plants by PCR、qPCR、 Southern blot analysis.Southern blot analysis confirmed that GmTLPl gene had been integrated into the genome of transgenic tobacco with three or four copies.5. The over-expression and anti-expression transgenic lines displayed the reverse symptoms after TMV inoculation. Comparing to wild type plants, the over-expression transgenic plants showed faster disease development of Tobacco mosaic virus, whereas the anti-expression transgenic plants showed delaying disease development of tobacco mosaic virus.