Research On The Xylanase Inhibitor In Grains

The distribution of the xylanase and xylanase inhibitor protein in different cereal seeds were researched,also we cloned the taxi-Ⅰgene which encoding the protein of xylanase inhibitor obtained from wheat variety Zhengmai 366.Then we transformed it into Escherichia coli and Pichia pastoris to express for investigate the characterizations of the recombinant xylanase inhibitor.The results are as follows:(1) In our study, nine common cereal seeds including wheat, barley, rye, green wheat, buckwheat, oats, corn, rice and sorghum are selected as experimental material to study their endogenous xylanase and xylanase inhibitor protein activity. The results show that endogenous xylanase activity is higher in corn but lower in other cereals, and the xylanase inhibitor protein activity are higher in wheat, rye, green wheat and buckwheat but lower in barley and oats; the xylanase inhibitor protein activity cannot be detected in corn, rice and sorghum. Further more, we detected the endogenous xylanase and xylanase inhibitor protein in wheat, rye and green wheat with higher xylanase inhibitor activity. The results showed that endogenous xylanase activity are higher in embryos than in seed coat and endosperm, between which there are similar results. The xylanase inhibitor protein activities are equal between the embryo and endosperm, but much higher than in the seed coat.(2) The xylanse inhibitor protein gene taxi-Ⅰwas cloned: The RNA was extracted from the kernel’s immature embryo of Zhengmai 366 and the taxi-Ⅰgene was cloned using the RT-PCR technology.(3) The taxi-Ⅰwas expressed in Escherichia coli: The taxi-Ⅰwas inserted into the expression vector of pET30a(+) and transformed into E.coli BL21(DE3) .But for some reasons taxi-Ⅰgene was not expressed successfully in Escherichia coli. And then taxi-Ⅰgene was inserted into the expression vector of pPIC9K with theα-factor signal sequence and transformed into Picha pastoris GS115. By SDS-PAGE and activity detection we screened low expression of TAXI-Ⅰx ylanase inhibitor strains.