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Synthesis of Cry1Ca Pesticidal Gene, Expression in E.coli and Transgenic Indica Rice Development

Author: LinZhiMin
Tutor: WangFeng
School: Fujian Normal University
Course: Biochemistry and Molecular Biology
Keywords: Rice Bt crylCa Agrobacterium-mediated transformation Lepidoptera Synthetic
CLC: S511
Type: Master's thesis
Year: 2010
Downloads: 35
Quote: 1
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Abstract


On the basis of the same amino acid sequence of the original gene , according to Rice codon bias and frequency of use, optimization, transformation cry1Ca gene to improve the GC content , remove affect the sequence of the mRNA structure factors of instability , increase in before and after cry1Ca gene conducive to a leader sequence for expression in plants and termination of a recognition sequence to add BamHI restriction sites at the ends of the gene , etc. , and the ultimate synthesis of full-length gene sequence 1912bp . PET - 28bca , prokaryotic expression vector build crylCa gene was induced by IPTG E. coli characteristics of the fusion protein with six histidine-tagged fusion protein expression and inclusion body purification using Ni metal ion chelating affinity chromatography on denatured dissolved the fusion protein was purified by size for the purpose 67KDa protein . After refolding immunize New Zealand white rabbits were obtained highly specific polyclonal antibody titer of 1:10000 . Double T-DNA build cry1Ca gene the plant expression carrier pCDMARCA , Agrobacterium transformation of rice Minghui 86 by Southern blot hybridization analysis showed that cry1Ca gene has been integrated into the genome of rice chromosome ELISA results also showed that transgenic expression in rice leaves to express accounted leaves 0.01-0.28 % of the total soluble protein . Survey of insect resistance in the field under natural conditions and, ultimately, the 33 the insect resistance turn cry1Ca gene T0 generation materials .

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