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Cloning 1Dx5 Gene from the Xinjiang Wheat Variety and Constructing Its Expression Vector

Author: LuXiaoYu
Tutor: ZuoJianBing
School: Xinjiang Normal University
Course: Botany
Keywords: Wheat High Molecular Weight Glutenin Gene Cloning Sequence Analysis Genetic Transformation
CLC: S512.1
Type: Master's thesis
Year: 2011
Downloads: 13
Quote: 1
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Abstract


The compositions and numbers of the high molecular weight glutenin subunit (HMW-GS) would directly affect and determine the flour baking quality in wheat seed. 1Dx5 gene was cloned from the main Spring Wheat Cultivars in Xinjiang (Triticun aestivum L., AABBDD) named xinchun 19 has the subunit 5, Analysed by subunit nucleic acid sequence, protein sequence, the structural differences and molecular evolution relations. The plant expression vector was also constructed and transformated into Arabidopsis.Cloning and sequence analysis: in this paper, HMW-GS 5 subunit gene from Xinchun 19 was cloned and named 1Dx5-XJ19. The sequencing results showed that: 1Dx5-XJ19 gene was 4658bp, containing 2544bp code region, encoding 848 amino acids, promoter are 1780bp, containing promoter and enhancer sequences characteristic sequence, 3 ’end contains 334bp. 1Dx5-XJ19 similarity achieved 99% with X12928 which from bread wheat. Two amino acids had been replaced compared with CAA31395 come from bread wheat.One was located at the signal peptide area and the other was in middle repeat area. It contained four cysteines. Through the secondary structure prediction of amino acids, highβ-turn content to be found. These indicated that the cloned gene was the high quality subunit gene.Establishment of plant expression vector: Insertting correct gene into pMON530, after CaMV 35S promoter, so the plant expression vector was constructed named pMON-1Dx5-XJ19. By PCR and restriction enzyme digestion analysis, the insert fragment was about 4.7kb, contains double promoters.Transformation of Arabidopsis thaliana: Obtaining engineering strain includes target gene. Target gene was transformed to Arabidopsis thaliana by floral dip transformation method. The T0 generation seeds were sowned on 1/2MS medium containing 50mg/L kanamycin for screening transgenic seedlings. Transformants were analysed by PCR. The results showed that 1Dx5-XJ19 gene had been inserted into the Arabidopsis genome. Whether the target gene is expressed in Arabidopsis would be our next research work.The result of this study not only builds a technology platform for crops in Xinjiang, also has the important theory value and the application significant in the triticum aestivum variety’s molecular breeding.

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