Detection And Analysis Of RGA And HCBT Resistance Related Genes In Wheat Leaf Rust Resistance Near-isogenic Lines

Wheat leaf rust disease, caused by Puccinia triticina, is serious damage to the wheatyield, quality and food safety. Therefore, exploring and using of wheat leaf rust resistancegenes are significance for resistance breeding and disease controling. In this study, wedesign primers according to the conservative structure domains of two kinds clonedresistance genes homologous sequences (RGA), STK and NBS-LRR, and a defense relatedgene, hydroxyl cinnamoyl/benzene armour acyltransferase (HCBT), to test the resistancegene types in50wheat leaf rust resistance near isogenic lines (NILs) and Thatcher. Then,we analysed the expression differences in different organs of12NILs. The results are asfollows:1. We designed7pairs primers, according to the conservative structure domains ofSTK, LZ-NBS-LRR, TIR-NBS-LRR, NBS-LRR and HCBT-like plant disease resistancerelated genes, to test the resistance gene types in51wheat NILs. The results showed that2~4kinds of resistance genes detected in each line.2. STK-like genes were amplified by primer CDS2in the cDNA of roots, stems,leaves in TcLr1, TcLr9, TcLr10, TcLr34and Thatcher, stems, leaves of TcLr19, TcLr21andTcLr45, and leaves of TcLr24, TcLr38, at seedling stage. And existed in all inspectedorgans except the roots of TcLr1, TcLr9, TcLr10, TcLr19, TcLr24, TcLr26, TcLr45andThatcher at adult stage. LZ-NBS-LRR-like genes were amplified by primer CDS3only inthe roots of TcLr1, TcLr9, TcLr10, TcLr21and Thatcher at seedling stage, and wereamplified in all the organs of TcLr1, TcLr29and Thatcher, roots, stems, leaves, and flagleaves of TcLr21and TcLr24, roots and stems of TcLr10and TcLr19, roots of TcLr9,TcLr26and TcLr38at adult stage. NBS-LRR-like genes were amplified by primer CDS6inthe stems and leaves of11varieties except TcLr29at seedling stage, but could be amplifiedin all the inspection organs of TcLr1, TcLr10, TcLr19, TcLr26and TcLr45, roots and stemsof TcLr9, TcLr21, TcLr24, TcLr34, TcLr38and Thatcher at adult stage. HCBT-like genesamplified by primer CDS7existed in the root, stem and leaf of Thatcher, stems and leavesof TcLr9and TcLr45, stems of TcLr1, TcLr10, TcLr19, TcLr21and TcLr38at seedlingstage. And could be amplified in all the tested organs of TcLr1, TcLr24, TcLr26, TcLr29,TcLr34, TcLr38, TcLr45and Thatcher, stems of TcLr10, TcLr21and TcLr29at adult stage. 3. The bands amplified by STK-like primer CDS2in the seedling leaves of TcLr1,TcLr10, TcLr19and Thatcher, were cloned and sequenced the length of bands were829bp,865bp,829bp and865bp, respectively. And the biggest open reading frames (ORF) were274,286,274and274amino acids, respectively. The results of BLASTn showed all thegenes were highly homologous with the receptor protein kinase sequences of commonwheat. All the genes had five subregions of STK type of resistance genes, so belonged tothe STK type resistance genes.4. The bands amplified by LZ-NBS-LRR-like primer CDS3in the seedling roots ofTcLr1, TcLr10and Thatcher, were all cloned and sequenced to be898bp. And the biggestopen reading frames (ORF) were174amino acids. The results of BLASTn showed all thegenes were100%similar to wheat resistance to strip rust gene Yr10mRNA and theLZ-NBS-LRR-like resistance related gene of Aegliops tauschii. All the genes had both theNBS disease-resistant gene structure domain (kinase2a,3a) and HD hydrophobic structuredomain.5. The bands amplified by NBS-LRR-like primer CDS6in the adult leaves of TcLr19,TcLr45and Thatcher, were all cloned and sequenced to be703bp. And the biggest openreading frames (ORF) were203amino acids. The results of BLASTn showed all the geneswere100%similar to the NBS-LRR-like resistance related gene of Aegliops tauschii. Allthe genes had the resistance related conservative structure domains.6. The bands amplified by Hydroxy cinnamoyl/benzene armour acyltransferase(HCBT)-like primers CDS7in the seedling stems of TcLr1, TcLr9, TcLr10, TcLr45andThatcher, were all cloned and sequenced to be723bp. And the biggest open reading frames(ORF) were224amino acids. The results of BLASTn showed all the genes were100%similar to the HCBT-like resistance related gene of Aegliops tauschi. All the genes had theplants acyltransferase corresponding catalytic subregion HXXXDG.7. The result of Southern blot showed the LZ-NBS-LRR gene cloned from TcLr1waslow copy in12wheat NILs. The NBS-LRR and HCBT genes in TcLr45and Thatcher wereinduced by through the semi-quantitative analysis, and were expressed highest in36h and24h, respectively. Furthermore, the expression amount of the two genes in the combinationof incompatibilities was higher than in compatibilities.