Effect Of CymMV And ORSV Infection On Leaf Protein Expression In Phalaenopsis Amabilis&Cloning And Prokaryotic Expression Of CymMV TGB1and TGB2Gene

Phalaenopsis of orchid Phalaenopsis is a single stem plants. The flower has a graceful shape and beautiful color. It is known for diverse names such as "empress orchid" and the Carter Leah blue, Vanda and Dendrobium are known as the four large ornamental orchid, has high ornamental value and economic value. However, Phalaenopsis is susceptible to infection. The most common infections are from Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Susceptible leaves appear to have chlorotic stripes, saggy gray spots or necrotic ring spot and other symptoms. When infected, the plant grows undesirably, the flower is small and less, and the flowering is shortened. Often, the complex infection endangers aggressively, reduce the economic value of the orchid and reduce the ornamental value. Therefore, study of the mechanism of interaction between Phalaenopsis and viruses for the development of breeding disease resistance Phalaenopsis has important significance.This study mainly used the proteomics approach to study of Phalaenopsis and virus interactions protein changes. On one hand, using two-dimensional electrophoresis and mass spectrometry study of susceptible protein change in phalaenopsis, preliminarily discussed the molecular mechanism of virus infection response in Phalaenopsis from protein level and sought crucial proteins related to plant virus and host interaction mechanism. On the other hand, using the CymMV TGB1(Ttriple Gene Block, TGB), TGB2gene to construct prokaryotic expression vector and express GST-TGB1, GST-TGB2fusion protein, then through the GST Pull-down to find the protein of Phalaenopsis interaction with it. Finally, the study provides a theoretical bases and an effective way to breed resistant varieties of Phalaenopsis.Using two-dimensional electrophoresis, the leaves were separated into healthy and susceptible (CymMV/ORSV) Phalaenopsis leaf total with protein and had discovered27differential proteins. Identification by mass spectrometry MALDI-TOF-MS analysis found that these proteins are closely related with disease resistance, stress response, transcriptional regulation, energy metabolism, protein modification, and other biological functions. Among them, we speculate that resistance protein RGC2, Beta-cyanoalanine synthase, ATP sulfurylase, Glutamate decarboxylase isozyme2, and peptide deformylase may play important roles in protein interaction during the virus infection process.According to the genomic sequence of Cymbidium mosaic virus (CymMV) in Genbank (Accession No. AB197937), specific primers were designed for amplifying the TGB1and TGB2gene, respectively. By reverse transcription-polymerase chain reaction (RT-PCR), the TGBl and TGB2genes were amplified from CymMV-infected Phalaenopsis leaves. Both fragments were then inserted into pGEX-4T3vector for prokaryotic expression. The constructed expression vectors were transformed into E. coli BL21that was induced by IPTG to express the target proteins. SDS-PAGE and Western blot analysis confirmed that the expressed proteins were GST-fused CymMV TGB1and TGB2proteins. Expression of TGB1and TGB2will help to prepare the antibodies for their further functional analysis.