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Cloning, Expression of Citrus Maxima Limonins Glycosyltransferase Gene and Construction of Plant Expression Vector

Author: MaZuo
Tutor: LiMingYang
School: Southwestern University
Course: Cell Biology
Keywords: Limonoids glycosyltransferase Clone Sequence analysis The prokaryotic expression Plant expression vector
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 92
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Abstract

Limonoids is one of the main reasons that cause citrus bitter, the citrus bitter taste fresh and processed citrus fruit quality have significant adverse impact. In addition, many pharmacologically active anti-tumor, anti-inflammatory, analgesic, antifeedant limonoids. Limonoids have these features, so the demand for limonoids increasing, but the bitter taste of the existence of limiting the intake of it. Limonoids glycosides with water-soluble and non-bitter taste and other characteristics, is similar to its biological activity and limonoids. Thus as alternative food additives, have good market prospects. On the basis of previous studies, the study from the Liangping pomelo amplified a limonoids glycosyltransferase gene CmLGT. Has constructed prokaryotic expression vector and plant CmLGT gene expression vector, the main findings of the study are as follows: 1. The limonoids glycosyltransferase enzyme gene CmLGT amplification and encoded protein nature analysis limonoids in GenBank glycosyltransferase (LGT) of the nucleotide sequence of the gene were designed and synthesized specific primers. In order the Liangping pomelo adult tree leaves total RNA as a template, limonoids glucosyl transferase gene, full-length coding region was amplified by PCR and bioinformatics methods CmLGT the nature and structure of the gene encoding protein characteristics analysis, Results Showing: it encodes 511 amino acids, the molecular weight of about 57.467 KD, an isoelectric point of 6.01 to 21 phosphorylation sites, without the signal peptide. CmLGT protein secondary structure by the α-helix (helix, 28.18%), extended strand (extended strand, 22.11%), random the curl (random coil, 49.71%). NCBI on their conserved domain analysis found CmLGT gene encoding the protein has a GT family characteristics. Genes 2.CmLGT the prokaryotic expression vector system for the optimization of the construction and expression of the purpose cloned into the prokaryotic expression vector pET-28a (), construct the gene fusion expression vector pET28a-CmLGT transformed into E. coli BL21 (DE3) induced expression of purpose fusion protein of approximately 62 kD in size, optimal conditions (4 h, 37 ℃ and 1.0 mmol · L-1PTG), soluble fusion protein analysis found that the fusion protein form of inclusion bodies. Plant expression vectors constructed by double digestion, ligation, transformation target gene CmLGT connection to plants expression vector pCAMBIA2301g, transforming Agrobacterium tumefaciens LBA4404 using BamH Ⅰ and Sac Ⅰ double enzyme digestion and PCR validation was successfully constructed. target gene plant expression vector and transformed into Agrobacterium tumefaciens LBA4404. 4. import wild tobacco Nicotiana tabacum application Agrobacterium-mediated leaf disk containing limonoids the glycosyltransferase gene CmLGT plant expression vector resistance screening plants, GUS staining and PCR amplification test, nine transgenic tobacco plants.

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