Prokaryotic Expression And Antibody Preparation Of Wheat Cell Wall Invertase CWI-B1 And Function Analysis Of TaCWI-B1 Overexpression Wheat

Cell wall invertase?CWI?is considered to key invertase involved into plants growth and development,crop quality and yield,response to abiotic stress and pathogenic bacteria.Wheat?Triticum aestivuml?is the major crop,Few researches about the biological roles of the cell wall invertase in wheat growth and development,yield and quality traits were reported.Cao Rufei a doctor of our lab cloned TaCWI-B1 and constructed TaCWI-B1 overexpression vector drived by ubiquitin promoter.In order to uncover the important roles of CWI in wheat?Triticum aestivum?,We firstly prepared of polyclonal antibody of TaCWI-B1.Molecular identification,genetic analysis after obtained transgenic wheat with TaCWI-B1 by agrobacterium mediated transformation.The main outcomes are as below:?1?Build prokaryotic expression vector and prepared polyclonal antibody for Wheat?Triticum aestivum?cell wall invertase gene TaCWI-B1TaCWI-B1 is the cell wall invertase of wheat?Triticum aestivum?which encode a kind of hydrophilic protein after analysed by bioinformatics.The vector pET28a-TaCWI-B1 was build by inserting the coding region of TaCWI-B1 into expression vector,and transferred into BL21?DE3?.The induction conditions,including isopropyl?-D-1-thiogalactopyranoside?IPTG?concentration,temperature,and culture time,were optimized.It turned out that the best condition for the target protein production was 0.2 mmol/L of IPTG,and induction at 28?for 6 h.The His-tag fused TaCWI-B1protein was expressed in the form of inclusion body, purified by Ni-NTA Sefinose TM Resin Kit and the MALDI-TOF-MS analysis indicated it was really the cell wall invertae of wheat?Triticum aestivum?.After separation and purification target protein,the polyclonal antibody was prepared,its titer reached over 1:50000.The result of Western blotting demonstrated that the TaCWI-B1 polyclonal antibody could specifically recognize not only the recombined protein,but also the target protein from wheat?Triticum aestivum?.?2?Molecular detection transgenic wheat plants with TaCWI-B1 by agrobacterium mediated transformationWe got 12 positive transgenic wheat lines by specific PCR identification.After transfer to greenhouse,there were 11 plants survived and 10 plants with seeds.The number of transgenic copy of T₀ transgenic plants showed that all the tested plants were single copy number except one line T₀-4.Genetic analysis of T_0:1:1 of 6 lines show exogenous gene TaCWI-B1can stablely inherit to the next generation.All the plants mRNA relative expression were much more in different degrees than wild type except T₁-6-4,T₁-13-2 and T₁-13-3.Western blotting analysis show that the expression level of target protein was consistent with the target gene mRNA in transgenic plants.In this study,we will continue to detect and track the transgenic lines of homozygous transgenic,and to determine the biological effects of TaCWI-B1 gene in transgenic wheat.