Cloning And Functional Analysis Of TiAsp Gene From Thinopyrum Intermedium Induced By Blumeria Graminis F. Sp. Tritici

Wheat is an important food crops in China. Its yield is related to national food security. Therefore, raising output of wheat plays a key role in agricultural development. However, wheat powdery mildew, as a main disease, restricted the development of wheat production and quality. Because there are so many kinds of wheat powdery mildew races and these races are known to mutate rapidly, the wheat crops containing a single resistance gene would easily lose resistance in the field. Therefore, finding and cloning the multiple resistance genes are important for wheat breeding. In previous research, a wheat-Thinopyrum alien addition line with high resistance to powdery mildew SN6306 which originated from the intergeneric hybridization between YN15 and Thinopyrum intermedium(Host) Nevski was obtained. Basic results are shown below:(1)Cloning and functional analysis of TiAspPrevious transcriptome analyses showed that TiAsp were mainly expressed in SN6306, without expression in YN15. Then, TiAsp gene which encoding aspartic proteinase was further studied. Through PCR amplification, the full length cDNA 1272 bp of TiAsp are obtained. Its open reading frame(ORF) is 1272 bp and encodes a peptide of 423 amino acids. Certain softwares were used to analyze the signal peptide, the results showed TiAsp contained signal peptide and its cleavage site is located between the 16 th and 17 th amino acid residues. ASP proteins maybe secreted proteins.An ASP protein in SN6306 is similar to the ASP proteins in other species, such as Triticum urartu and Aegliops tauschii. The result of BSMV-VIGS showed that conidial powder of powdery mildew was appeared on the leaves of SN6306 when TiAsp gene was silenced. In fact, under the normal conditions, SN6306 were resistant to wheat powdery mildew when TiAsp was not silenced. These data suggested that TiAsp is involved in the response to Blumeria graminis f. sp. tritici(Bgt). The result of the qRT-PCR indicated that the expression level of TiAsp was up-regulated under the induction of methyl jasmonate(MeJA) or salicylic acid(SA). Furthermore, the expression level of TiAsp was down-regulated at first and then up-regulated. The highest peak of TiAsp expression emerged at 60 h under the induction of MeJA, and the highest peak of TiAsp expression emerged at 24 h under the induction of SA. Menawhile, the expression level of TiAsp was up-regulated by more than 30 times compared with the non-induced controls for MeJA and more than 4 times compared with the non-induced controls for SA. These data indicated that Ti Asp is involved in the response to methyl jasmonate or salicylic acid.(2)On the basis of Ubi promoter and pMBX17-YUC10 vector, a pMUbi-Tiasp vector was constructed. Through the particle bombardment method, TiAsp gene was translated into wheat cultivar Bobwhite callus and transgenic plants have been obtained. Meanwhile, overexpression vector pUbi-TiAsp was constructed and has been transformed into wheat cultivar Y158 and K199.(3)Expression of the TiAsp gene in Pichia pastoris expression system for studying the function of ASP proteinA vector pPIC9K-TiASP for expressed in P. pastoris was constructed, and further study will be conducted through the P. pastoris expression system., The TiAsp will be induced to express in yeast by methanol, and the induced proteins will secreted into the cell culture supernatant. The recombinant proteins will be purified in order to analyze its function.