Abstract：Cell wall invertase(CWI) is the key regulator which forms sugar concentration gradient between source and sink by catalyzing sucrose hydrolysis and promotes sucrose into sink. In order to uncover the important roles of CWI in wheat (Triticum aestivum), bioinformatics were utilized to analyse the protein properties and structure map of wheat TaCWI-B1. The prokaryotic expression vector pET28a-TaCWI-B1 was constructed by inserting the coding region of TaCWI-B1 into pET28a, and transformed into Escherichia coli strain BL21(DE3). The induction conditions, including isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, culture temperature and induction time, were optimized, and the recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified. After identifying the recombinant protein by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the polyclonal antibody was prepared, its titer was measured by enzyme-linked immunosorbent assay (ELISA) and its specificity was analyzed by Western blot. The results showed that the full-length cDNA of TaCWI-B1 was 1 845 bp and this gene had a ORF of 1 776 bp and encoded a protein of 591 amino acid with molecular mass 65.92 kD and pI 9.26. The analysis based on software of Protscale and Swiss-model showed that TaCWI-B1 was a hydrophilic protein with a structure of N-acetyl glucosamine outside, and it was a major component of cell walls. The prokaryotic expression showed that TaCWI-B1 was expressed abundantly as inclusion bodies, and the optimum 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-B1 protein was purified by Ni-NTA SefinoseTM Resin Kit and the MALDI-TOF-MS analysis showed that the similarity between recombinant protein and TaCWI-B1 was 99.992%, which indicated it was really the cell wall invertae of wheat. ELISA showed that the serum antibody titer of 2 TaCWI-B1 immunized rabbits reached over 1∶50 000. The result of Western blot demonstrated that the TaCWI-B1 polyclonal antibody could specifically recognize not only the recombined protein, but also the target protein from wheat. These results provide basic data for further investigation of TaCWI-B1 gene functions.
［1］Roitsch T, González M C. Function and regulation of plant invertases: sweet sensations[J]. Trends in Plant Science, 2004, 9(12):606-613. ［2］Sturm A, Tang G Q. The sucrose-cleaving enzymes of plants are crucial for development, growth and carbon partitioning[J]. Trends in Plant Science, 1997, 4(10):401-407. ［3］Hirose T, Takano M, Terao T. Cell Wall Invertase in Developing Rice Caryopsis: Molecular Cloning of OsCIN1 and Analysis of its Expression in Relation to its Role in Grain Filling[J]. Plant and Cell Physiology, 2002, 43(4):452-459. ［4］Carlson S J, Shanker S. Chourey P S. A point mutation at the Miniature1 seed locus reduces levels of the encoded protein, but not its mRNA, in maize［J］. Molecular Genetics and Genomics, 2000, 263(2): 367-373. ［5］Cho J I, Lee S K, Ko S, et al. Molecular cloning and expression analysis of the cell-wall invertase gene family in rice ( Oryza sativa, L.)[J]. Plant Cell Reports, 2005, 24(4):225-36. ［6］Lammens W, Roy K L, Laere A V, et al. Crystal structures of Arabidopsis thaliana cell-wall invertase mutants in complex with sucrose[J]. Journal of Molecular Biology, 2008, 377(2):378-85. ［7］Weber H, Buchner P, Borisjuk L, et al. Sucrose metabolism during cotyledon development of Vicia faba L. is controlled by the concerted action of both sucrose-phosphate synthase and sucrose synthase: expression patterns, metabolic regulation and implications for seed development[J]. Plant Journal for Cell & Molecular Biology, 1996, 9(6):841-850. ［8］Weschke W, Panitz R, Gubatz S, et al. The role of invertases and hexose transporters in controlling sugar ratios in maternal and filial tissues of barley caryopses during early development[J]. Plant Journal, 2003, 33(2):395–411. ［9］Sreenivasulu N, Altschmied L, Radchuk V, et al. Transcript profiles and deduced changes of metabolic pathways in maternal and filial tissues of developing barley grains[J]. Plant Journal, 2004, 37(4):539-553. ［10］Ishimaru T, Hirose T, Matsuda T, et al. Expression Patterns of Genes Encoding Carbohydrate-metabolizing Enzymes and their Relationship to Grain Filling in Rice (Oryza sativa L.): Comparison of Caryopses Located at Different Positions in a Panicle[J]. Plant and Cell Physiology, 2005, 46(4):620-628. ［11］Wang ET, Wang JJ, Zhu XD, et al. Control of rice grain-filling and yield by a gene with a potential signature of domestication[J]. Nature Genetics, 2008, 40(11):1370-1374. ［12］Godt D E, Roitsch T. Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism[J]. Plant Physiology, 1997, 115(1):273-82. ［13］Sherson S M, Alford H L, Forbes S M, et al. Roles of cell wall invertase and monosaccharide transporter in the growth and development of Arabidopsis[J]. Journal of Experimental Botany, 2003, 54(382):525-531. ［14］Tang G Q, Lüscher M, Sturm A. Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning[J]. Plant Cell, 1999, 11(2):177-189. ［15］Miller M E, Chourey P S. The Maize Invertase-Deficient miniature-1 Seed Mutation Is Associated with Aberrant Pedicel and Endosperm Development[J]. Plant Cell, 1992, 4(3):297-305. ［16］Wang ET, Xu X, Zhang L, et al. Duplication and independent selection of cell-wall invertase genes GIF1 and OsCIN1 during rice evolution and domestication[J]. BMC Evolutionary Biology, 2010, 10(1):1-13. ［17］Giura A, Saulescu N N. Chromosomal location of genes controlling grain size in a large grained selection of wheat ( Triticum astivum, L.)[J]. Euphytica, 1996, 89(1):77-80. ［18］Ma DY, Yan J, He Z, et al. Characterization of a cell wall invertase gene TaCwi - A1, on common wheat chromosome 2A and development of functional markers[J]. Molecular Breeding, 2012, 29(1):43-52. [19] Lingle SE, Dunlap JR. Sucrose Metabolism in Netted Muskmelon Fruit during Development［J］. Plant physiol, 1987, 84（2）: 386-389.
