Transformed EuCHIT1 Gene In Wheat(Triticum Aestivum) Enhances Resistance To Stripe Rust And Leaf Rust

Wheat(Triticum aestivum),the world's largest cultivated crop in both total production and cultivated areas.Rust disease is a severe fungal disease in wheat species that results in decreased yield and quality.Nowadays,choosing the wheats resistant to rust diseases has become the main way to reduce the disease loss.It is important to explore a new way of improving rust resistance with introducing exogenous resistance genes in wheat breeding.In this work,the Agrobacterium-mediated transformation method were adopted to introduce EuCHIT1(Eucommia ulmoides chitinase1)gene into wheat variety “Guizi3”,“Mingxian169”,“AVS” and “SY95-71”,the trangenic plants were obtained according to the result of ?-glucuronidase histochemical staining and PCR identification.The chitinase activities in the transgenic wheat plants and wild-type were measured,the resistance to rust and protective enzymes activities and relative expression level of pathogenesis-related protein genes were measured..The main results were summarized as follows.1,The Agrobacterium tumefaciens mediated shoot apical meristem transformation methodIn this research,the Agrobacterium tumefaciens mediated shoot apical meristem transformation method were adopted to introduce EuCHIT1(Eucommia ulmoides chitinase1)gene into wheat variety “Guizi3”,“Mingxian169”,“AVS” and “SY95-71” and trangenic plants were obtained according to the result of ?-glucuronidase histochemical staining and PCR identification.2,Effect of EuCHIT1 gene expression on wheat agronomic traitsThe transgenic wheat plants and wild-type(“Guizi3”)were grown in field at the transgenic plant facility.There was no obvious difference between the wild-type and transgenic plants by comparing the agronomic traits such as the height,length and weight.The results suggested that the expression EuCHIT1 in wheat plants did not have significant negative effects on the performance of the transgenic lines in the field.3,Effect of EuCHIT1 gene expression on wheat chitinase activitiesThe chitinase activities in transgenic wheat plants and wild type(“Guizi3”)were mensurated.The results indicated that chitinase activities in transgenic plants were 42.21% higher than that in wild-type(2340.72 U/g(FW)),reached to 3328.63 U/g(FW).The results suggested that expression of the transgene EuCHIT1 in wheat plants increased chitinase activities.4,Effect of EuCHIT1 gene expression on wheat resistance to stripe rustAfter inoculating with the fungus,the lesions of the transgenic plants and wild type leaf explants appeared for 14 days and 5 days respectively.The transgenic plants delayed the occurrance of stripe rust disease 9 d compared to the wild-type.The identification of the resistance to stripe rust on wheat leaves 14 d after inoculating showed that the transgenic plants appeared high resistance,while the wild-type was moderately susceptibel.Additionally,the lengths of flag leaves with diseases in the transgenic wheats were shorter than those in the wild-type(4.50 vs 9.60 cm).5,Effect of EuCHIT1 gene expression on wheat resistance to leaf rustThe transgenic wheat plants and wild-type at the heading stage were inoculated with the fungus.The results showed that the average Maximum disease severity(MDS)in transgenic wheat(“Mingxian169”,“AVS” and “SY95-71”)were 62.00%,52.75% and 58.50% respectively,which were 35.4%,29.8% and 27.3% lower than in wild type.The average Area under the disease progress curve(AUDPC)in transgenic wheat(“Mingxian169”,“AvS” and “SY95-71”)were 374.50,307.12 and 344.75,which were 34.5%,40.7% and 41.1% lower than in wild type.The results suggested that expression of the transgene EuCHIT1 in wheat plants enhanced the disease resistance.6,Effect of EuCHIT1 gene expression on wheat protective enzyme activitiesIn our study,the catalase(CAT),super-oxide dismutase(SOD)and peroxidase(POD)activities activities and the malondialdehyde(MDA)content in wild type and transgenic plants(“Guizi3”)were determined before and after inoculation with race CYR32 for 7 d.Before inoculation of the pathogen,the POD activities in transgenic plants were 19.60% higher than that in wild type,reached to 40.96 nmol/g;SOD and CAT activities had not significant difference between wild type and transgenic plants.MDA contents in transgenic plants were 25.61% lower than that in wild type,reached to 5.84 nmol/g;However,after inoculating with the fungus for 7 days,the CAT,SOD and POD activities were 211.91 U/g,448.37 U/g and 81.30 U/g(FW)in the transgenic plants and 159.95 U/g(FW),294.38 U/g(FW)and 37.87 U/g(FW)in the wild-type plants.This showed that the CAT,SOD and POD activities in the transgenic wheats were 32.48,49.76 and 114.68%,respectively,higher than that in the wild type,The results also indicated that the MDA content in transgenic plants was 8.69 nmol/g,which was 29.23% lower than 12.28 nmol/g in the wild type.The results suggested that expression of the transgene EuCHIT1 in wheat plants enhanced the antioxidant capacity,and reduced damage to stripe rust.7,Effect of EuCHIT1 gene expression on wheat pathogenesis-related protein genesThe relative expression levels of pathogenesis-related protein genes in wild type and transgenic plants(“Guizi3”)were analyzed before and after inoculation with race CYR32 for 7 d,with wheat Cell division control protein(CDC)gene as an internal reference,The relative expression levels of Pathogenesis-related protein1 gene(PR-1),Pathogenesis-related protein2 gene(PR-2)and Pathogenesis-related protein5 gene(PR-5)in the transgenic plants were respectively 1.14,6.61 and 3.87 folds higher than those in the wild-type before inoculation of the pathogen.After inoculation with the fungus,the expression levels of PR-1,PR-2 and PR-5 in the transgenic plants was up-regulated 2.14,3.41 and 7.55 folds higher than the wild-type,respectively.The results suggested that transgenic plants increased relative expression level of pathogenesis-related protein genes,thereby enhanced the disease resistance of transgenic plants..