Analysis Of The Expression Of HvS/TPK Gene And Transformation Of Antisense-HvS/TPK Gene Into Triticum Aestivum-Haynaldia Villosa Translocation Line 92R137

Powdery mildew,caused by Erysiph graminis f.sp tritici,is one of serious fungal diseases of common wheat worldwide.Development and utilization of wheat cultivars is an efficient approach to control the disease.Pm21 gene,which was located on the short arm of chromosome 6V of H.viUosa,is a broad-spectrum and powerful powdery mildew resistance gene.Cao et al.(2006) cloned a candidate gene of Pm21,HvS/TPK,by using gene chip technique.To investigate the expression and the functions of HvS/TPK gene,we analyzed the expression characters of HvS/TPK gene,and transformed antisense-HvS/TPK gene into Triticum aestivum-Haynaldia villosa translocation Line 92R137.1.Analysis of HvS/TPK gene expressionThe expression characters of HvS/TPK induced by biotic or abiotic stresses treatments were studied.The results showed that the expression of HvS/TPK was upregulated by Ergsiphe greminis Dc.f.sp.tritici,and by ethephon(10mM) and H₂O₂(7mM) treatments, indicating that the expression of HvS/TPK gene may be associated with ethylene-dependent defense response and reactive oxygen species-mediated defense response.The expression level of HvS/TPK did not increase when inoculated with Fusarium graminearum,or treated by high temperature(40℃),high salinity(300mM NaCl) and 5mM salicylic acid solution. Additionally,the up-regulated expression of HvS/TPK gene was not only found in leaves, but also in spikes when they were inoculated with Ergsiphe graminis De.f.sp.tritici.These results indicated that HvS/TPK gene may not be associated with the response to Fusarium head blight,as well as to hot shock and salt stress. 2.Expression of HvS/TPK gene in E.coli.To further study the biological functions of HvS/TPK gene in resistance response to wheat powdery mildew,a prokaryotic expression vector pET32a-HvS/TPK was constructed, and the conditions of expression of the recombinant protein,His-HvS/TPK,were optimized. The maximum expression level of His-HvS/TPK was obtained when the concentration of ITPG,the inducing temperature and the inducing time were at 1.0～1.5mg/L,28℃and four hours,respectively.The molecular weight of His-HvS/TPK was approximately 55.0KDa, which was in consistent with our prediction.Total protein was extracted from bacteria,and the supernatant fluid and precipitate were used for SDS-PAGE eletrophoresis.The results showed that the His-HvS/TPK existed as an inclusion body.3.The bioinformatics research on HvS/TPK geneThe sequence of HvS/TPK gene and its encoding product were compared with other genes and proteins from barley,rice and arabidopsis by using online blast software provided by NCBI website.The results showed that there were many genes and proteins sharing high homology with HvS/TPK gene and its encoding product(identity between nucleotide sequences not less than 80%,identity between amino acid sequences not less than 50%).These homologous genes encode proteins containing a putative Ser/Thr kinase domain.Also,a putative kinase domain was predicted at the N-terminus of HvS/TPK(from 3 to 153) through the online conversed domain prediction tool at NCBI website.Such evidence demonstrated that HvS/TPK gene might be a member of a new subfamily of Ser/Thr kinase gene family.The predictive three-dimensional model of the kinase domain of HvS/TPK and its functional motifs and residues were obtained by using SWISS-MODEL online tools and Cn3D software provided by NCBI in this paper.The results showed that a motif HRDIKASNIL at locus 79-88,was putative catalytic loop;three motifs DFGLAKLLPP, ISTRV and GTLGYLAPE,located at 99 to 108,113 to 117 and 119 to 127 respectively, formed an activated loop;residues 28^Tyr,86^Asn,88^Leu and a motif SDF(98-100) formed a putative ATP binding pocket;and residues 35^Ser,83^Lys,115^Thr,116^Arg,118^Gly,120^leu and 121^Gly formed a putative substrate binding pocket. 4.Transformation of antlsense-HvS/TPK gene into 92R137One thousand two hundred and twenty-four immature embryos were bombarded with the pAHC-HvS/TPKa construct,and thirty-nine regeneration plants were obtained.These plants were tested by PCR with the primers of the interest gene,bar gene and ubi promoter, and 11 plants were proved to be transgenic plants.The frequency of transformation was 0.9%.