| Wheat sharp eyespot(Rhizoctonia cerealis van der Hoeven apud.Boerema & Verhoeven), caused by the soil-bome fungi(Rhizoctonia cerealis Vander Hoeven)and(Rhizoctonia solani Kuha), is one of the serious diseases in wheat growing regions in the world.The increasingly serious occurrence of wheat sharp eyespot made us unable to promote wheat production in China at present. It was necessary to carry out new researches and strategies.For this,research advances in its etiology,occurrence,damage,as well as resistant germplasm screening were reviewed and some advice on further study was also proposed.In the wheat genetic engineering research against wheat sharp eyespot,one of the major obstacles is the lack of immunization or stable high resistant gene sources.Therefore,the screen of resistance gene to wheat sharp eyespot as an effective defense gene is great significance.Plant activate downstream defensive reaction against pathogens,and inspired a series of defense-induced expression gene.In this study,experimental material is wheat(Triticum aestivum)Shannong0431 which is resistant plants.Pathogen-induced downstream PR genes were screened by semi-quantitative RT-PCR method with inhibition experiments in vitro,then we identified the inhibitory effect of wheat sharp eyespot by the downstream defensive genes screened as resistance genes resources against wheat sharp eyespot.Our main conclusions as follows:(1)Screeningβ-1,3-glucanase gene(DQ-GLU)induced by wheat sharp eyespot.The transcript level of DQ-GLU was up-regulated at 6h after inoculation of R.cerealis,and declined at 24h in Shannong0431,which is resistant plants.It was not expressed in the normal plants,which were not inoculated by R.cerealis.And the low expression of transcription happened in the susceptible plants of Wenmai6.So we speculated DQ-GLU gene is involved in the the reaction of wheat sharp eyespot resistance.(2)Construction of DQ-GLU gene prokaryotic expression vector,the expression of recombinant induced by IPTG,refolding in vitro and purification,inhibition activity assay in vitro,the coding region of the DQ-GLU gene cloned into the expression vector pGEX-4T-1 and transformed into E.coli BL21(DE3).IPTG induced the E.coli BL21(DE3)transformed with the GLU-pGEX plasmid to express the recombinant fusion protein GST-GLU.The results showed that most of the recombinant protein was expressed in the form of inclusion bodies.To purify the target protein,various buffers with different pH levels and different denaturants were tried,and we established a set of simple and practical refolding system.The purified glucanase showed a broad-spectrum inhibition activity in vitro against hyphal growth of several pathogenic fungi: Rhizoctonia solani(rice sheath blight),R.cerealis(wheat sharp eyespot),Alternaria alternata (tobacco brown spot),and Phytophthora capsici(capsicum epidemic diseases).(3)Cloning chitinase RC7 gene and constructed prokaryotic expression vector,the expression of recombinant induced by IPTG,refolding in vitro and purification,inhibition activity assay in vitro.RC7 gene was inserted into the prokaryotic expression vector pGEX-4T-1,expressed the fusion protein of GST-RC7 successfully in Escherichia coli BL21(DE3).Further analysis showed that the fusion protein of GST-RC7 almost entirely in the form of inclusion bodies.Chitinase activity assay showed that RC7 of prokaryotic expression protein was refolded in vitro successfully. Inhibition activity assay in vitro showed that the chitinase inhibited the growth of the mycelium against R.solani of the strongest inhibition,followed by R.cerealis,Fusarium graminearum, VerticilliuIll dahliae,A.alternata.(4)constructed transformation vectors pUGLU and pURC7,bombarded embryos of Yangmai 12 by biolistic particle method.332 regenerated plants with resistance to Bialaphos were achieved on the selection medium by Bialaphos.46 transgenic plants with Nos genes were identified by PCR assay in the To generation,we screened out of 16 resistance plants in the 46 transgenic plants with disease-resistant identification.(5)The distribution and disease-resistant identification of Rs-AFP2 was characterized.Southern blot results show that there is one copy Rs-AFP2 in transgenic wheat and Rs-AFP2 has stably integrated into the chromosomes of wheat.Resistance assay results show that the positive plants appear higher resistance(Maturity index for the level 0),compared to the control plants and molecular testing negative(Maturity index for the level 3～4).Therefore,Rs-AFP2 could be used as a potential gene for improving broad resistance spectrum in wheat breeding programs.
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