CDNA Library Construction Of TcLr24Induced By Puccinia Triticina And CDNA Cloning And Analysis Of Gene GMP Synthetase

Wheat leaf rust, caused by Puccinia triticina, is one important worldwide disease onwheat, constantly discovering new resistance genes and developing new disease-resistantvarieties are significance to the prevention and treatment of wheat leaf rust disease.Cloning and related functional analysis of resistant gene will provide important basis forutilization of disease-resistant varieties.As one of the few widely effective genes conferring resistance to leaf rust in wheat,Lr24was transferred into the wheat genome from Agropyron elongatum. To date, it hasgreat potential to be used for wheat production. Our cDNA library was constructed bywheat near isogenic line TcLr24leaves induced by avirulent isolate of P. triticina. Also weused RACE (rapid amplification cDNA ends) technique to obtain full length sequence ofthe specific expression gene in TcLr24. Prokaryotic expression vector with GPMsynthetase gene was constructed, and the fusion proteins was produced by E. coliexpression system. The main results were as follows:1. The leaves of TcLr24were harvested at0h,6h,12h,18h,24h,36h,48h,60h,72hand96h, respectively, after inoculation with THTT biotype of Puccinia triticina. TotalRNA was extracted from inoculated wheat leaves, then equal amount of which then weremixed as reverse transcription template. A compatible cDNA library was constructed usingIn-Fusion SMARTerTM Directional cDNA Library Construction Kit. The muber ofcloning of this library was2.1×106. The clones of the library had an average insert size of1.0kb, which ranged form0.5kb to1.5kb. ATP binding protein, putative cyclin-T1familyprotein, histone deacetylase HDAC3, fibrous sheath CABYR-binding protein auxintransport protein etc were blasted to have homologus with that in other plant in the30clones randomly piked.2. A cDNA sequence of the aimed GMP synthetase gene was cloned from TcLr24byusing RACE technique based on the target fragment R27amplified by RNF. Total RNA ofTcLr24was isolated and the first strand of cDNA was synthed. Specific primer of5′RACEwas designed based on the target sequence. The750bp fragment was amplified fromTcLr24cDNA by touchdown protocel. The sequenced fragment was then assembled to thetarget band successfully and1475bp full-length cDNA was acquired. Full-length gene primer was designed,1048bp and1816bp sequence was amplified from cDNA and gDNAof TcLr24, respectively, the gene include4introns and5extrons. It was verified further inTcLr24leaves induced by P. triticina with RT-PCR. The sequence had a length of1475bp,including a1038bp complete open reading frame encoding345amino acid, a221bp5′untranslated terminal region (5′UTR), a216bp3′untranslated terminal region (3′UTR)and29bp polyA tails. The deduced amino acids of R27protein contained GMP synthase Cterminal domain and asparagine synthase domain. Phylogenetic tree indicated that R27might share a common ancestor with Aegilops tauschii, Hordeum vulgare, and has farrelationship with Sorghum bicolor. The molecular weight of the protein is38.64kDa, is ahydrophilic protein. This gene was located on wheat1B chromosome by chromatidpositioning.3. After inserting R27open reading frame into pGEM-T easy vecter, the recombinantplasmid R27-T was digested with restriction enzyme BamHâ… å'ŒXhoâ… . Digested fragmentwere directly inserted into the expression vector pET-28(+) digested with the same doubleenzymes. The prokaryotic expression vector with the target gene R27-pET-28a wassuccessfully constructed, and then was transformed into E.coli Transetta (DE3). Themolecular weight of the fusion proteins with about38kDa was obtained.