Isolation And Pathogenicity Analysis Of The Tengu Gene From Wheat Blue Dwarf Phytoplasma

Wheat blue dwarf disease (WBD) is the first reported wheat phytoplasma disease in China and has not been reported abroad. It is disseminated by Psammotettix striatus L.. The disease is widely distributed in northwest China's arid and semi-arid late-maturing winter wheat region. Once the outbreak, a huge loss of production of winter wheat will be caused. Thus, it is extremly important to study and prevent wheat blue dwarf disease.It is difficult to culture phytoplasma in vitro and phytoplasma has a low level of content in host, thus the study of this pathogenic microorganism is restricted. In this thesis, the tengu gene of wheat blue dwarf phytoplasma was isolated and analysed using bioinformatics. Prokaryotic expression vector was constructed. In addition, plant virus expression vectors of the wheat blue dwarf tengu gene,the immunodominant membrane protein (imp) gene and the hemolysin (hlyC) gene were constructed. This three genes were expressed in Nicotiana benthamiana. The results of the studies are listed as follows:(1) Isolation the tengu gene from wheat blue dwarf phytoplasma: The gene was amplified using specific primers from total DNA of wheat blue dwarf phytoplasma samples which were collected from Hancheng, Shannxi Province. Comparison analysis of the tengu gene of wheat blue dwarf phytoplasma and onion yellows phytoplasma (OY) showed that the tengu gene wheat blue dwarf phytoplasma was 213 bp(GenBank accession number:JF923816), and encoded 70 amino acids. The wheat blue dwarf phytoplasma tengu gene had the homology of 93.42% with aster yellows phytoplasma (AY) and 95.77% with onion yellows phytoplasma (OY).(2) Molecular structure characteristics of the tengu gene: The signal peptide cleavage site of the calculation using SignalP 3.0 software showed that the cleavage site was between the 32 alanine and the 33 aspartate at N-terminal of the tengu protein, so the tengu mature protein had only 38 amino acids. Hydrophobicity analysis showed that hydrophobicity of the tengu protein was -0.270, and -1.308 after the signal peptide was removed. The mature protein was a hydrophilic protein. Secondary structure prediction showed that the tengu protein was mainly constructed by threeα-helix folds and oneβ-sheet unit. Possible transmembrane region for the tengu protein showed the protein was a significant transmembrane protein, the transmembrane region was between the 11th and 30th amino acids in N-terminus.(3) The prokaryotic expression vector of pET30-tengu was constructed by adding restriction sites. This established the foundation for further study of the tengu gene research at protein level.(4) The plant virus expression vectors of pGR107-tengu, pGR107-imp and pGR107-hlyC were constructed and transformed into agrobacterium. Then Nicotiana benthamiana leaves were infected with agrobacterium. At transcriptional level the genes can be detected 7 days later. All the tobaccos showed symptom of mosaic which was potato virus X (PVX) infection symptom, this indicated that the infection was successful. Compared to other genes, there were 2 of 15 infected Nicotiana benthamianas which had more branches and stunt than others. This indicated that expression of the wheat blue dwarf phytoplasma tengu gene in Nicotiana benthamiana can cause Nicotiana benthamiana shorter, branches increased.