Cloning And Function Analysis Of Apple Alternaria Blotch Resistant Gene Mal D 1

As a serious fungal disease,Apple Alternaria blotch has a severe effect on Chinese apple industry every year.At present,researches about this disease at home and abroad are mainly focused on control methods,pathogen identification and resistant varieties breeding.While the mechanism of disease resistance remains unclear.Recently,it has been reported that,after Alternaria blotch,apple scab or fire blight infection,the Mal d 1 gene can be induced to express and participate in defense responses in apple leaves.However,the specific role of Mal d 1 gene in defense responses requires further excavation.Regarding the importance of identifying the function of apple resistant genes,we cloned apple disease resistant gene Mal d 1,preliminarily accomplished its function identification and explained its expression features during infection process.All these work contributes to theoretical basis and technical support for further study in genetically developing and breeding new apple resistant varieties.In this study,Mal d 1 gene was cloned from "Orin" apple leaves.Gene functions were obtained by bioinformatics analysis,Real-time PCR,transgenic plant materials disease resistance identification and fusion protein induced expression.The main results are as follows:1.Bioinformatics analysis showed that the open reading frame(ORF)of Mal d 1 gene is 480 bp long and consists of 2 exons and 1 intron.The coding sequence is annotated to encode a protein of 159 amino acids.The molecular weight of Mal d 1 protein is about 17.62 kD,which was predicted to be composed of 19 different kinds of amino acids.The isoelectric point,aliphatic index andinstability index of Mal d 1 protein are 5.68,82.26 and 31.80,respectively.Mal d 1 protein is predicted to be localized in cytoplasm by PSORT Prediction tool.According to the predicted three-dimensional structure of Mal d 1 protein,the numbers of amino acids that form ?-helix,?-sheet and random coil structure are 35,48 and 76,respectively.2.Real-time PCR data showed measurable expression of Mal d 1 in apple leaves,flowers,fruit peels and pulps,while the expression level varied.Mal d 1 can be detected in fruit peels during all stages of fruit development,with a initially increased and then decreased expression trend.Mal d 1 expression was detectable in the leaves of all different varieties,with a relatively high expression level in resistant varieties.Under the condition of Alternaria blotch inoculation on apple leaves,Mal d 1 was significantly upregulated compared to the plants without inoculation.These results suggest that Mal d 1 gene is involved in the interaction between plant and pathogen.3.Mal d 1 overexpressed transgenic callus,which was obtained by agrobacterium-mediated transformation method using PRI101-AN as the expression vector,showed a greatly enhanced resistance to Alternaria blotch.4.The recombinant vector pCold-Mal d 1 was constructed by ligating Mal d 1 with the prokaryotic expression vector pCold.Then the recombinant plasmid pCold-Mal d 1 was transformed into Escherichia coli BL21(DE3)strain.SDS-PAGE analysis showed that Mal d 1 protein was stable and highly expressed after cold-induction.The molecular mass of Mal d 1 fusion protein was about 22.4 KDa.The protein which was purified by nickel column could be used for immunohistochemistry and Western Blot.