Cloning And Expression Of The Xylanase Inhibitor Gene From Wheat

The dynamic changes of xylanase and xylanase inhibitor activity in wheat at different growth stage were analyzed,and the gene xip encoding the mature protein of xylanase inhibitor obtained from Wheat variety Zhengmai 366 was cloned,and then overexpressed in Escherichia coli and Pichia pastoris to investigate the characterizations of the recombinant xylanase inhibitor.There were four parts in this study:(1) The research of dynamic changes of xylanase and xylanase inhibitor activity in wheat at different growth stage: The dynamic changes of xylanase and xylanase inhibitor activity in wheat at different growth stage were analyzed using field-planted Zhengmai 366、Lankao Aizao 8 and Yumai 49 as materials. The results showed that there was no difference in xylanase and xylanase inhibitor activity between the three wheat varieties, and that no significant changes of xylanase activity whether in leaves or kernels were observed during different growth stages, indicating that xylanase might exist in wheat leaves and kernels as a basal level. The xylanase inhibitor activity in leaves increased as a whole from seedling stage, through jointing stage to heading stage, but significantly decreased when entering grain filling-maturing stage, while the xylanase inhibitor activity in kernels increased continuously with the maturation of kernels.(2) The xylanse inhibitor protein gene xip was cloned: The RNA was extracted from the kernel’s immature embryo of Zhengmai 366, The xip gene (GenBank number: GU318402)was cloned using the RT-PCR technology.(3) The xip was expressed in Escherichia coli: The xip was inserted into the expression vector of pET21a(+) and transformed into E.coli BL21(DE3) which was expressed successfully in Escherichia coli by inducing with IPTG.(4) The xip was expressed in Pichia pastoris: The xip gene with theα-factor signal sequence was inserted into the expression vector of pPIC9K and transformed into Picha pastoris GS115 .