Over-expression Of Xylanase With High Specific Activity From Streptomyces Olivaceoviridis A1 In Plants

Xylan is hetero-polysaccharides consisting of a polymer of β-1,4-linked xylose units substituted with side groups. It is the major component of crops, and has been found to be anti-nutritional in mono-gastric animals, particularly in poultry. The enzymatic hydrolysis of xylan is accomplished by the action of endo-1,4-β-xylanase (EC3.2.1.8) and β -D-xylosidase (EC3.2.1.37). Xylanase hydrolyzes 1,4-β -xylosidic linkages to generate small oligosaccharides. In general, high level production of xylanase has been pursued with microbial fermentation. However, transgenic plants can give us substitution. Some works have shown that microbial xylanase can be successfully expressed in tobacco, barley and rice, although the activity and enzyme content can not be sufficient for industrial purpose. In this project, we firstly expressed the xylanase with high specific activity from Streptomyces olivaceoviridis A1 in transgenic plants, in order to harvest the transgenic plants with high xylanase activity.The gene encoding XYNB with high specific activity from Streptomyces olivaceoviridis Al was transformed into tobacco under the control of an efficient double 35S promoter and AMV enhancer. The integration of the xynB in the genome of tobacco was confirmed by PCR. Moreover, RT-PCR, ELISA, SDS-PAGE and Western blot analysis confirmed the expression of XYNB enzyme. In tobacco, high levels of the 21 kD protein, which was as same as that of original xylanase (XYNB), was synthesized in cytoplasm or targeted to the intercellular space by means of the potato proteinase inhibitor II signal peptide. The recombinant xylanase was accumulated to the level of up to 6% of total soluble leaf proteins. And in transgenic leaf tissues, xylanse activity up to 170 IU/g fresh leaf (23 IU.mg-1 total protein) was observed. The xylanase expressed in transgenic tobacco can be stable after treated in different temperatures. Furthermore, growth and development of the transgenic tobacco was not affected by the expression of enzyme, and T₁ plants showed the genetic stability.Potato expressing xylanase constitutively and tuber-specifically was firstly constructed. Molecular analysis show that high levels of the 21 kD and glycosylated 31kD protein, was synthesized in cytoplasm and targeted to the intercellular space by signal peptide respectively. The expressed xylanase was accumulated to the level of up to 5% of total soluble leaf proteins in transgenic potato. Xylanse activity up to 90 IU/g fresh leaf (10 IU.mg-1 total protein) was observed, while transgenic potato tuber activity was up to 13 IU/g fresh tuber.The transgenic potato transformed by vector in which the xynB gene led by tuber specific Patatin promoter can be induced to produce tuber. RT-PCR, Western blot and enzymatic activity analysis proved that the XYNB was specially expressed in tuber. The special xylanase activity was up to 10IU/g fresh tuber.The xylanase expressed constitutively in transgenic potato can be stable after treated in 60℃ and 70℃ water baths. Xylanase activity did not decrease when propagated 3-4 generations, which...