Cloning Of Germin Gene From Wheat And The Construction Of Plant Expression Vector Of Germin Gene

The development of the new gene cloning and genetic transformation techniques recently has opened the way of engineering for improving fungal resistance of crops. Fungal pathogens of crop plants cause extensive economic damage to farmers by reducing the yield .Generally the transgenes for fungal resistance are targeted towards reinforcement of plant cell walls or inhibiting the growth of fungal pathogens by producing antifungal proteins. Several pathogenic fungi secrete high amounts of oxalate which is the essential pathogenic factor. Germin in wheat is a manganese containing homohexamer with oxalate oxidase activities, which generates II2O2 from the oxidative breakdown of oxalate thereby playing a significant role in plant development and defense.Introduction of oxalate oxidase gene in susceptible crop plants thus represents a promising approach for engineering of disease resistance in plants against oxalate secreting fungal pathogen.The purpose of this research is to achieve a germin gene from wheat and construe its plant expressing vector. The main results are as follows:1. Cloning and sequence analysis of germin gene from wheatGermin in wheat have an higher oxalate oxidase activity. A pair of primers were designed according to the germin gf-2.8 gene sequence reported in GenBank.Germin gene was isolated from the genomic DNA of wheat "Xiangmail3" by PCR amplification. The PCR products were cloned into pDive vector and sequenced. The results of comparative analysis on NCBI-BLAST indicated that the cloned fragment contained 812 nucleotides and shared a homology of 90% with the reported sequence of germin gf-2.8 gene while its amino acid sequence shared a homology of 96%,A majority of the nine differences between and among the two sequences involve equivalent amino acids. The three conserved histidine residues with a conserved glutamate residue fits the active site of the homology model of oxalate oxidase.2. Construction of plant binary expression vector of germin geneThe germin gene fragment cut with BamH I from intermediated vector pDG28 was inserted into binary vector pBI121 cut with the same enzyme to formrecombinants which was selected by plaque PCR .Then recombinants were cut with Hind III and migrated as doublets in agarose gels. But the short fragments of the forward inserted recombinants (named as pBG128) had 968 nucleotides while that of the reverse inserted recombinants had 1,567 nucleotides. Then plant expression vector pBG128 contains a germin gene ,a Nptll gene as the selectable marker and a B -gluceronidase gene as the reporter gene. The pBG128 was introduced into Agrobacterium tumefaciens strain LBA4404 through the way of electrotransformation. And LBA4404 with pBG128 were identified by plaque PCR. 3. Transient expression of GUS geneThe explants derived from rapeseed line westar was infected for 20min by Agrobacterium tumefaciens LBA4404 with pBG128 of OD600 about 0.3-0.4, which were induced and activated in medium in presence of acetosyringohe for 3h. The explants were cocultivated with A. tumefaciens for 2 days about 28 "C in darkness and followed by P-glucuronidase (GUS) activity assay using lmg/ml X-Gluc. The X-Gluc gived a blue precipitate at the sil,e of enzyme activity in co-cultured explants under dissecting microscope. Transient expression of GUS gene was successful in co-cultured rapeseed explants.