Transformation And Function Analysis Of Powdery Mildew Resistance-related Genes From Triticum Aestivum And Haynaldia Villosa

Powdery mildew,caused by Erysiphe graminis f.sp tritici,is one of the most serious diseases of common wheat in China and many other countries in the world.A broadspectrum and powerful powdery mildew resistant gene,Pm21,was identified and introduced from H.villosa into common wheat through chromosome translocation,and located on the short arm of chromosome 6V in Cytogenetics Institute,Nanjing Agricultural University.The cloning of Pm21 will be critical for improving powdery mildew resistance level by genetic transformation.Several powdery mildew resistance-related and Pm21 candidate genes have been isolated from wheat or H.villosa.To characterize their potential functions by overexpression assays,it is necessary to establish a stable and high efficient transformation system.In the present research,the wheat tissue culture and regeneration system was first optimized.Immature embryo from twenty wheat cultivars was used as explant for tissue culture.Different hormone combinations and concentrations were applied for callus induction,differentiation and regeneration.The result showed that MXD₂ medium containing 500 mg/L Casein hydrolysate,100 mg/L Proline,100mg/L Glutamine and 40g/L Maltose was the best for callus inducion.As for callus differentiation,1/2MX medium containing 0.5mg/L IAA and 1mg/L ZT had the highest callus differentiation frequency of 83.0%.For plant regeneration,1/2MX containing 0.5mg/L IAA and 0.5mg/L MET was the best medium and obtained high frequency of surviving plant.Significant differences of response to tissue culture among wheat genotypes were observed.Eight varieties including Yangmai₁58,Nannong₉918,Jimai₂0,and Ningmai₉ et al.should be most suitable for immature embryo culture and regeneration and could be used for genetic transformation. Three different explant resources,immature embryo,mature embryo and inflorescence of Yangmai158,were further compared for their callus induction and plant regenerate ability to determine their potential use in wheat tissue culture.Callus induction mediums with different hormone concentration were used to study their effects on the frequency of callus induction,embryo germination,callus differentiation and quality,average weight,growth rate etc.Suitable plant regeneration culture system has been established.Five resistance-related genes,wheat thaumantin-like protein gene(Ta-Tlp),wheat antisense Mlo gene(aTa-Mlo),H.villosa glutathione geductase gene(Hv-GR) and H. villosa serine/threonine kinase gene(Hv-S/TPK),were transformed by microprojectile-bombardment using powdery mildew susceptible wheat cultivars as receptor.Transgenic plants were identified by molecular genetic analysis and evaluated for their powdery mildew resistance.Four genes,Ta-Tlp,aTa-Mlo,Hv-GR and Hv-S/TPK,were constructed into plant expression vectors driven by the strong ubi promoter and the bar gene as selection marker.Plasmids were transformed into immature embryo-derived calli.After two rounds of herbicide bialaphos selection and regeneration,herbicide-resistant plants were identified.PCR,Southern blot,and RT-PCR analysis confirmed the presence of the transgenes in the host genome.Totally 21,45,23 and 97 transgenic plants for the above four genes were obtained respectively.Average transformation efficiency was as high as 1.32%.Most transformants had more than one trangene copy.The transgenic plants of T₀, T₁,and T₂ generations were evaluated for their disease resistance.The Ta-Tlp transgenic plants of T₀,T₁ and T₂ generations showed delayed disease development of powdery mildew,while no distinct improved scab resistance was observed compared with the susceptible receptor.Partial overexpressing antisense Ta-Mlo transplants showed moderate susceptible or high resistance of powdery mildew.Overexpression of Hv-GR enhanced the resistance to powdery mildew and induced transcript accumulation of other pathogenesis-related genes such as PR-1a and PR-5 through changing the redox state of inner glutathione. Overexpression of Hv-S/TPK transplants showed high powdery mildew resistance throughout the growing period,and produce only a few hypersensitive response spots and sporadic piles of spore on the leaves.Ta-LRR₂ gene was transformed into wheat using a co-transformation method,in which target gene Ta-LRR₂ and the bar gene were in separated vectors(pBI-LRR₂ and pAHC25, repectively) and co-transformed simutaneously.Eighty-nine Ta-LRR₂ transgenic plants were identified by PCR,Southern blot,and RT-PCR analysis.Average transgenic efficiency was about 1.85%.The transgenic plants of T₀ and T₁ generations were evaluated for powdery mildew resistance.All transgenic plants showed high powdery mildew resistance at the seedling stage,while some hypersensitive response spots were observed on the leaves. At the adult stage,the transplants showed moderate powdery mildew resistance,and the disease symptom was found on the bottom leaves of the plants.Ninty-two transplants,in which contained the target gene but no marker gene,were furthermore identified.This could escape the biosafety problems by selection marker gene and should be more useful in wheat improvement for disease resistance.