新疆小麦1Dx5基因的分离克隆及表达载体构建
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摘要
小麦种子中的高分子量麦谷蛋白亚基(high molecular weight glutenin subunit,HMW-GS)组成和数量直接影响着小麦面包烘烤品质的优劣。本文以新疆主栽春小麦(Triticun aestivum L.,AABBDD)品种新春19为实验材料,分离克隆了优质5亚基基因,并分析了它与国外面包小麦中优质5亚基基因间核酸序列、蛋白质序列以及结构差异和分子进化关系,进而构建了植物表达载体和拟南芥的遗传转化。
主要研究结果如下:
基因克隆与序列分析:利用PCR技术从新春19中分离克隆5亚基基因,所克隆基因命名为1Dx5-XJ19。测序结果表明:1Dx5-XJ19基因全长4658bp,编码区有2544bp,编码848个氨基酸,启动子及5’UTR有1780bp,含有启动子特征序列和增强子序列,3’端有334bp。所克隆基因与数据库中提交从国外优质面包小麦中克隆的5亚基基因,序列号为X12928相似性达到99%。由1Dx5-XJ19所推导的氨基酸序列与数据库中面包小麦氨基酸(序列号CAA31395)有两处氨基酸的替换,分别位于信号肽区和中间重复区,氨基酸中含有4个半胱氨酸,两个连续的终止子。通过对推到氨基酸二级结构预测发现β-转角含量高,结果表明所克隆基因是1Dx5优质亚基基因。
植物表达载体构建:将目的基因插入表达载体pMON530 CaMV 35S启动子之后,构建植物表达载体pMON-1Dx5-XJ19,经PCR检测、酶切鉴定,插入的目的条带大小与克隆片段大小一致,均约为4.7Kb左右,表明目的基因已有效构建到表达载体中,所构建的表达载体含有CaMV 35S启动子和1Dx5-XJ19自身启动子序列。
拟南芥中转化:构建含有目的基因的工程菌株GV-pMON-1Dx5-XJ19,采用花序浸泡法转化拟南芥(Arabidopsis thaliana L.),收获T_0代种子播种于含有50mg/L卡那霉素的1/2MS培养基上进行抗性筛选,采用PCR技术对抗性幼苗进行检测,结果表明:1Dx5-XJ19基因已插入到拟南芥基因组中,对于目的基因能否在拟南芥种子中成功表达,表达蛋白与新春19中5亚基SDS-PAGE迁移率是否相同,有待于实验继续进行。
本文的研究结果不仅为新疆农作物优质基因的操作搭建了技术平台,而且为新疆小麦品种的分子改良育种奠定了坚实的理论和应用基础。
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 T_0 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.
主要研究结果如下:
基因克隆与序列分析:利用PCR技术从新春19中分离克隆5亚基基因,所克隆基因命名为1Dx5-XJ19。测序结果表明:1Dx5-XJ19基因全长4658bp,编码区有2544bp,编码848个氨基酸,启动子及5’UTR有1780bp,含有启动子特征序列和增强子序列,3’端有334bp。所克隆基因与数据库中提交从国外优质面包小麦中克隆的5亚基基因,序列号为X12928相似性达到99%。由1Dx5-XJ19所推导的氨基酸序列与数据库中面包小麦氨基酸(序列号CAA31395)有两处氨基酸的替换,分别位于信号肽区和中间重复区,氨基酸中含有4个半胱氨酸,两个连续的终止子。通过对推到氨基酸二级结构预测发现β-转角含量高,结果表明所克隆基因是1Dx5优质亚基基因。
植物表达载体构建:将目的基因插入表达载体pMON530 CaMV 35S启动子之后,构建植物表达载体pMON-1Dx5-XJ19,经PCR检测、酶切鉴定,插入的目的条带大小与克隆片段大小一致,均约为4.7Kb左右,表明目的基因已有效构建到表达载体中,所构建的表达载体含有CaMV 35S启动子和1Dx5-XJ19自身启动子序列。
拟南芥中转化:构建含有目的基因的工程菌株GV-pMON-1Dx5-XJ19,采用花序浸泡法转化拟南芥(Arabidopsis thaliana L.),收获T_0代种子播种于含有50mg/L卡那霉素的1/2MS培养基上进行抗性筛选,采用PCR技术对抗性幼苗进行检测,结果表明:1Dx5-XJ19基因已插入到拟南芥基因组中,对于目的基因能否在拟南芥种子中成功表达,表达蛋白与新春19中5亚基SDS-PAGE迁移率是否相同,有待于实验继续进行。
本文的研究结果不仅为新疆农作物优质基因的操作搭建了技术平台,而且为新疆小麦品种的分子改良育种奠定了坚实的理论和应用基础。
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 T_0 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.
引文
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