果实特异性2A12启动子克隆与桃ACO基因反义表达载体构建
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摘要
桃(Prunus persica)属蔷薇科多年生落叶果树,是典型的呼吸跃变型果实,达到生理成熟的果实采后很快出现呼吸高峰,迅速变软,严重影响其销售与储运。乙烯是跃变型成熟果实的催熟激素,跃变型果实自我催化乙烯合成反应启始后,合成大量乙烯,ACC氧化酶(ACO又称乙烯形成酶EFE)是乙烯形成过程中的最后一个酶,起着关键的作用。而利用果实特异型启动子可以启动外源基因在受体植物果实中高效表达的特点,研究并克隆番茄果实特异性启动子和ACO基因,并结合反义RNA基因技术抑制ACO基因的表达,就能够抑制桃果实的乙烯合成,从根本上改变桃果实成熟后迅速软化的特性,对于大幅度延长桃储藏时间和增加经济效益,培育桃抗软化新品种,将具有重要理论和实践意义。
本研究以成熟的白粉桃果实为材料,运用基因克隆技术,克隆番茄2A12果实特异型启动子和桃ACO基因,构建由2A12启动子调控的桃ACO反义基因植物表达载体,为下一步建立桃高效遗传转化体系奠定实验基础。本实验的主要研究结果如下:
1.利用植物总RNA提取试剂盒成功提取了高质量的白粉桃果实总RNA,反转录成cDNA第一链,根据GenBank中已发布的桃ACO基因序列(登录号:AF319166)设计一对特异性引物进行RT-PCR扩增后,获得目的片段,经测序分析ACO基因为1246bp序列,比对后发现核苷酸和氨基酸的同源性分别为99%和98%,表明正确的克隆了桃ACO基因,并将克隆载体命名为pMD-ACO。
2.用CTAB法提取番茄基因组DNA,根据GeneBank(No: M87659.1)已登录的2A11启动子序列设计一对特异性引物进行PCR扩增,获得了869bp的2A12片段大小,该序列与已发表的2A11启动子(No: M87659.1)的核苷酸序列比对其同源性高达100%,初步表明成功的克隆了2A12启动子,并将克隆载体命名为pMD-2A。
3.分别用限制性内切酶SacⅠ和BamHⅠ双酶切pMD-ACO克隆载体和pBI121表达载体。将pBI121载体的GUS A片段切下来,ACO基因片段反向插入到了pBI121载体中,构建成中间表达载体pBI-anti-ACO。用限制性内切酶HindⅢ和BamHⅠ分别双酶切pMD-2A克隆载体和pBI121表达载体。将pBI121载体的CaMV 35S片段切下来,2A12启动子片段正向插入到了pBI121载体中,构建成植物表达载体pBI-2A。用SacⅠ和BamHⅠ双酶切中间表达载体pBI-anti-ACO和植物表达载体pBI-2A,将切下来的反向ACO片段插入到pBI-2A中,构建成pBI-2A-ACO植物表达载体。并进行了PCR和双酶切鉴定。
Peach belongs to Rosaceae, perennial deciduous fruit tree, which is a typically climacteric fruit, When the fruit is physiologically mature, a climacteric peak appears soon after the post harvest period, which leads to softening rapidly and seriously impacts on their sales and logistics. Ethylene is a ripening hormone of mature climacteric fruits. A large amount of ethylene is produced after climacteric fruits starting the self-catalytic ethylene synthesis. ACC oxidase (ACO also called ethylene-forming enzyme EFE) which is the last enzyme in the formation of ethylene plays a key role. Fruit-specific promoters can activate the high-level expression of foreign gene in the fruit of receptors plants. Then, researching and cloning tomato fruit-specific promoter and ACC oxidase gene, combining with antisense RNA gene technology inhibits the expression of ACC oxidase gene in order to reduce the ethylene biosynthesis of peach. In the end, the purpose of delaying fruit softening can be achieved. It is hopeful to fundamentally change the Characteristic of peach fruit ripening rapidly, and nurture new types of anti-softening, which substantially increase the storage time and the economic benefit of peache. It is very important for theoretical and practical significance to promote the faster industry development of peach in our country.
The study, with mature Baifen peach as the materials, took advantage of Cloning technology to get the fruit specific promoter 2A12 in tomato and ACO gene in peach, and then constructed the antisense ACO gene expression vector regulated by promoter 2A12, laying the foundation for establishing an efficient system of genetic transformation of peach. The results of this study were as follows:
1. High-quality RNA was extracted successfully by useing plant total RNA extraction kit, reverse transcripted into the first strand cDNA, according to ACO gene sequence of peach (accession number: AF319166) published in GenBank, a pair of specific primers were designed for RT-PCR amplification to get the fragment. Sequence analysis showed that ACO gene is a 1246bp full-length sequence. Compared with GenBank landed ACO gene (AF319166) nucleotide sequence and amino acid sequence, the homology were separately 99% and 98%, indicating that ACO gene of peach was successfully cloned, named as pMD-ACO.
2.Tomato genomic DNA was extracted by the method of CTAB. According to 2A11 promoter (accession number: AF319166) published in GenBank, a pair of specific primers were designed for PCR amplification, and 869bp of 2A12 fragment was obtained. Sequence analysis showed that the compared with the published sequence of the 2A11 promoter (No:M87659.1), the corresponding nucleotide sequence homology was up to 100%, which indicated 2A12 promoter successfully was cloned, and named as pMD-2A.
3. pMD-ACO cloning vector and the pBI121 expression vector were digestioned respectively by SacⅠand BamHⅠrestriction endonucleases. GUS A fragment of the pBI121 vector was cut down, and then ACO gene fragment was reversely inserted into pBI121 vector to construct intermediate expression vector pBI-anti-ACO. pMD-2A cloning vector and the pBI121 expression vector were digestioned respectively by HindⅢand BamHⅠrestriction endonucleases. When CaMV 35S fragment was cut down from pBI121 vector, 2A11 promoter fragment was inserted into the pBI121 vector, and constructed the plant expression vector pBI-2A. With SacⅠa nd BamHⅠdigesting intermediate vector pBI-anti-ACO and the plant expression vector pBI-2A, ACO fragment cut down was reversely inserted into pBI-2A, and constructed the plant expression vector pBI-2A-ACO after PCR and digestion identification, which proved the vectors were constructed successfully, and the vectors can be used to transfer into the plant of peach.
本研究以成熟的白粉桃果实为材料,运用基因克隆技术,克隆番茄2A12果实特异型启动子和桃ACO基因,构建由2A12启动子调控的桃ACO反义基因植物表达载体,为下一步建立桃高效遗传转化体系奠定实验基础。本实验的主要研究结果如下:
1.利用植物总RNA提取试剂盒成功提取了高质量的白粉桃果实总RNA,反转录成cDNA第一链,根据GenBank中已发布的桃ACO基因序列(登录号:AF319166)设计一对特异性引物进行RT-PCR扩增后,获得目的片段,经测序分析ACO基因为1246bp序列,比对后发现核苷酸和氨基酸的同源性分别为99%和98%,表明正确的克隆了桃ACO基因,并将克隆载体命名为pMD-ACO。
2.用CTAB法提取番茄基因组DNA,根据GeneBank(No: M87659.1)已登录的2A11启动子序列设计一对特异性引物进行PCR扩增,获得了869bp的2A12片段大小,该序列与已发表的2A11启动子(No: M87659.1)的核苷酸序列比对其同源性高达100%,初步表明成功的克隆了2A12启动子,并将克隆载体命名为pMD-2A。
3.分别用限制性内切酶SacⅠ和BamHⅠ双酶切pMD-ACO克隆载体和pBI121表达载体。将pBI121载体的GUS A片段切下来,ACO基因片段反向插入到了pBI121载体中,构建成中间表达载体pBI-anti-ACO。用限制性内切酶HindⅢ和BamHⅠ分别双酶切pMD-2A克隆载体和pBI121表达载体。将pBI121载体的CaMV 35S片段切下来,2A12启动子片段正向插入到了pBI121载体中,构建成植物表达载体pBI-2A。用SacⅠ和BamHⅠ双酶切中间表达载体pBI-anti-ACO和植物表达载体pBI-2A,将切下来的反向ACO片段插入到pBI-2A中,构建成pBI-2A-ACO植物表达载体。并进行了PCR和双酶切鉴定。
Peach belongs to Rosaceae, perennial deciduous fruit tree, which is a typically climacteric fruit, When the fruit is physiologically mature, a climacteric peak appears soon after the post harvest period, which leads to softening rapidly and seriously impacts on their sales and logistics. Ethylene is a ripening hormone of mature climacteric fruits. A large amount of ethylene is produced after climacteric fruits starting the self-catalytic ethylene synthesis. ACC oxidase (ACO also called ethylene-forming enzyme EFE) which is the last enzyme in the formation of ethylene plays a key role. Fruit-specific promoters can activate the high-level expression of foreign gene in the fruit of receptors plants. Then, researching and cloning tomato fruit-specific promoter and ACC oxidase gene, combining with antisense RNA gene technology inhibits the expression of ACC oxidase gene in order to reduce the ethylene biosynthesis of peach. In the end, the purpose of delaying fruit softening can be achieved. It is hopeful to fundamentally change the Characteristic of peach fruit ripening rapidly, and nurture new types of anti-softening, which substantially increase the storage time and the economic benefit of peache. It is very important for theoretical and practical significance to promote the faster industry development of peach in our country.
The study, with mature Baifen peach as the materials, took advantage of Cloning technology to get the fruit specific promoter 2A12 in tomato and ACO gene in peach, and then constructed the antisense ACO gene expression vector regulated by promoter 2A12, laying the foundation for establishing an efficient system of genetic transformation of peach. The results of this study were as follows:
1. High-quality RNA was extracted successfully by useing plant total RNA extraction kit, reverse transcripted into the first strand cDNA, according to ACO gene sequence of peach (accession number: AF319166) published in GenBank, a pair of specific primers were designed for RT-PCR amplification to get the fragment. Sequence analysis showed that ACO gene is a 1246bp full-length sequence. Compared with GenBank landed ACO gene (AF319166) nucleotide sequence and amino acid sequence, the homology were separately 99% and 98%, indicating that ACO gene of peach was successfully cloned, named as pMD-ACO.
2.Tomato genomic DNA was extracted by the method of CTAB. According to 2A11 promoter (accession number: AF319166) published in GenBank, a pair of specific primers were designed for PCR amplification, and 869bp of 2A12 fragment was obtained. Sequence analysis showed that the compared with the published sequence of the 2A11 promoter (No:M87659.1), the corresponding nucleotide sequence homology was up to 100%, which indicated 2A12 promoter successfully was cloned, and named as pMD-2A.
3. pMD-ACO cloning vector and the pBI121 expression vector were digestioned respectively by SacⅠand BamHⅠrestriction endonucleases. GUS A fragment of the pBI121 vector was cut down, and then ACO gene fragment was reversely inserted into pBI121 vector to construct intermediate expression vector pBI-anti-ACO. pMD-2A cloning vector and the pBI121 expression vector were digestioned respectively by HindⅢand BamHⅠrestriction endonucleases. When CaMV 35S fragment was cut down from pBI121 vector, 2A11 promoter fragment was inserted into the pBI121 vector, and constructed the plant expression vector pBI-2A. With SacⅠa nd BamHⅠdigesting intermediate vector pBI-anti-ACO and the plant expression vector pBI-2A, ACO fragment cut down was reversely inserted into pBI-2A, and constructed the plant expression vector pBI-2A-ACO after PCR and digestion identification, which proved the vectors were constructed successfully, and the vectors can be used to transfer into the plant of peach.
引文
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