Isolation And Pathogenicity Analysis Of Endo-1, 4-Beta-Glucanase Gene From Wheat Blue Dwarf Phytoplasma

Wheat Blue Dwarf was caused by phytoplasma and widely distributed in late ripe winter wheat varieties of semiarid and arid region areas in Northwest China, this disease was transmitted by Psammotettix striatus in a distinct way. Diseased wheat leaves show yellow, plant short, the leave turning thickening or hardening, crop failure or even no when all field diseased, it will be a big menace for the yield of wheat, so study the prevention of Wheat Blue Dwarf disease extremely important. Endo-1,4-beta-glucanase is a cellulose enzyme which was produced chiefly by fungi, bacteria, insect, animal and plant organization. The pathogenic bacteria can easily move when the enzyme act and soften the the cells of plants.In this paper, FrvX gene fragments of Wheat Blue Dwarf were amplified through PCR, and constructed prokaryotic expression and plant virus vector mediated transient expression. The results of the studies are listed as follows:(1) The isolation of FrvX gene of Wheat Blue Dwarf: the gene was amplified using specific oligonucleotides: FrvX-F/FrvX-R from wheat total DNA, the length of FrvX is 1 071 bp, G and C cotent of FrvX is 36.4 %, encoding 356 amino acids, GenBank accession numbers is FJ866635. The FrvX had the homology of 95.52 % with AY and 94.49 % with OY.(2) Use Prot–Scale,ANTHEPROT 5.0,TMHMM-2.0 software and 3D-PSSM web site predicate the hydrophilicityM, transmembrane, the secondary structure, the potential cleavage site of signal peptide and 3D structure of FrvX respectively. The result showed that molecular weight of FrvX was 38.7 KDa, its PI is 6.975, in the second structureα-helix,β-sheet and random coil are 45 %,26 % and 29 % respectively. There has a strong signals peptide cutting peptide in the 20th amino acid (glycine); FrvX has no transmembrane domains and it is a hydrophobic protein which was secreted outside the membrane.(3) The Prokaryotic Expression vector of pET30-FrvX is constructed, after IPTG induce, the transformed vector in host cell for expression, but no expection band. on the contrary, with the increasing time of culture, the quantity of host cell gradually decreasing, this may because the dissolution of FrvX to cell wall.(4 ) Construction plant virus expression vectors pgR107-FrvX and transformed into agrobacterium through electroporation, after inductived by phenolic compound (AS), tobacco leaves were infected with agrobacterium,7 days later, the gene can be detected at transcriptional level. According to statistical analysis, the infected recombinant plasmid can cause leaves above 45 % show winkle and yellow symptom, we infer FrvX is a pathogenetic gene of WBD.