Functional Analysis Of Aspartic Proteinase Gene TiAP1 From Thinopyrum Intermedium

Wheat is one of the most widely planting crops in our country.About a third of the population staple food is wheat in the world.However,wheat powdery mildew and fusarium head blight are the important limiting factor affecting wheat yield.Therefore,the current problems to be solved are finding new resistance genes and breading the wheat cultivars which have resistance to multiple diseases.In previous research,an aspartic acid protease gene TiAP1 was cloned from a wheat-Thinopyrum alien addition line SN6306 which originated from the intergeneric hybridization between YN15 and Thinopyrum intermedium(Host)Nevski.The main results are as follows:(1)Bioinformatics analysis of TiAP1 proteinAccording to predicting the transmembrane helices of TiAP1 protein,the protein has no transmembrane structure,and may not secreted proteins.At the same time,many physical and chemical characteristics of protein were analyzed.The result showed that the protein contains 506 amino acids residues and its theoretical p I is 5.6.TiAP1 contained signal peptide and its cleavage site is located between the 16 th and 17 th amino acid residues.Beyond that,the predicted secondary structure contains extended strand and random coil in high proportion,the model of third structure is stable.(2)The prokaryotic expression and function analysis of TiAP1 proteinOn the basis of pET-28 a vector,a pET-AP1 vector was constructed.E coil BL21(DE3)plysS cells harboring pET-28a/TiAP1 were induced by IPTG,and the expression protein was analyzed by SDS-PAGE.A band of approximately 45 kDa was present in BL21.In order to test the protein further,western blotting was done.The protein was purified using affinity chromatography.The result showed that expressed fusion protein was in the inclusions.The purified and renaturated TiAP1 protein inhibited spores germination of powdery mildew and F.Graminearum.(3)Membrane permeability analysis of TiAP1 proteinIn order to further determine the resistance mechanism of target protein,F.Graminearum hyphae and spores was incubated with different concentration TiAP1 protein.Then SYTOX Green was added to evaluate membrane integrity by fluorescence microscope.In the stock solution,there are hardly any fluorescent in the spores and hyphae.In the low concentration protein,there are little fluorescent.And in high concentration protein,the fluorescent hyphae and spores number increased obviously.These results declared that TiAP1 can increase the permeability of cell membrane.(4)The response to Pst DC3000 in transgenic ArabidopsisThe binary vector pROK2(preserved in our laboratory)was used to generate a plasmid for Arabidopsis transformation.When the transgenic A.Thaliana plants were grown at 2-3-leaf stage,Pst DC3000 was injected.To determinate the accumulation of the callus,the test of reactive oxygen and the number of dead cells,the Arabidopsis leaves were stained by aniline blue,trypan blue,DAB.There results showed that overexpression of TiAP1 in Arabidopsis increased the resistance to Pst DC3000.(5)The resistance identification of transgenic wheat to powdery mildewAfter the transgenic line incubated with the powdery mildew,the number of Bgt spores in transgenic lines leaf is less than the control both seedling stage and mature stage.In addition to that,the result of the qRT-PCR indicated that the expression level of TiAP1 was up-regulated under the induction of methyl jasmonate(MeJA).Furthermore,transcript levels of OPDA?PR10 and COL1 which mediate the JA-related defense pathway were substantially higher in transgenic plants than those in the control.Especially,the expression level of PR10 is increased 10 times higher than 0h.We presumed that the over-expression of TiAP1 stimulate JA signaling defense pathway.