紫花苜蓿MsZFN锌指蛋白基因启动子MsZPP的克隆与序列分析
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摘要
紫花苜蓿(Medicago sativa L.)是世界上分布最广的一种多年生豆科牧草,也是我国最重要的豆科牧草之一。它不仅产量高且营养丰富,被誉为“牧草之王”。传统的育种方法主要是通过杂交的手段获得新品种,需要的周期长,工作量大。将分子生物学的方法运用到了抗性育种工作中,可以缩短育种年限,提高育种效率。本试验主要研究的是一个锌指蛋白基因的启动子。锌指蛋白是真核生物基因组中最丰富的一类转录因子,这种蛋白质与Zn2+结合形成稳定的手指结构,在基因的表达调控、细胞分化、胚胎发育等生命过程中发挥重要作用。动物及人体内的锌指蛋白的功能已经有了较为深入的了解,近年来,通过不断的研究发现,锌指蛋白在植物中也发挥了重要作用,参与了植物抵御其生长过程中所经历的各种胁迫反应。
启动子(promoter)是RNA聚合酶能够识别并与之结合,从而起始基因转录的序列,作为转录水平上一种重要的调控元件,研究启动子,对我们了解生物的生长发育过程,探讨生物对其生长环境的适应机制以及更好地控制外源基因在转基因植物中的表达等具有重要意义。本研究取得的主要结果如下:
1.应用巢式PCR技术,克隆获得了锌指蛋白基因MsZFN的上游启动子区,大约1500bp左右,在Blast数据库上进行同源性比较,该基因为首次获得,与截形苜蓿7号染色体克隆mth2 - 36p20的全序列有88%的相似性。在GenBank上登录,登录号为FJ161979.1,命名为MsZPP。对获得的序列进行分析,发现了多个CAAT-box和TATA-box,TATA框具有将RNA聚合酶Ⅱ引导到正确的转录起始位点的功能,CAAT框主要控制着转录的起始频率,是调控转录速率的元件,与启动子的特异性无直接的关系。还发现了多个潜在的顺式作用元件,如与ABA诱导相关的顺式作用元件ABA响应元件(ABRE),与干旱诱导有关的MYB结合位点(MBS),与茉莉酸甲酯反应相关的顺式调控元件TGACG-motif,以及多个光响应元ACE,G-box,GT1-motif, I-box, SpI。
2.构建了启动子全长序列的表达载体并运用农杆菌介导的方法转化烟草,获得了转基因植株,PCR检测显示已将目的片段成功整合到烟草的基因组中。
3.根据预测的转录起始位点克隆不同长度的5’端缺失序列,并构建了用于转化的表达载体,运用农杆菌介导的方法转化烟草,获得了转基因植株,PCR检测显示已将目的片段成功整合到烟草的基因组中。
Alfalfa (Medicago sativa L.) is the world's most widely distributed perennial legume forage grass, and also is the most important one of the legumes forage grass. It is not only high yield but also nutrient-rich, so it is called the“king of grass”. Traditional breeding methods is mainly through the hybridization to abtain the new varieties. It required long cycle and heavy workload. Using molecular biology methods is the hot spot of current breeding research, especially applied to the resistance breeding work, it can shorten the breeding period, improve breeding efficiency.This study is mainly about a zinc finger protein gene’promoter. Zinc finger protein is the most rich protein in a class of transcription factors in eukaryotic genome, such proteins binding the Zn2 + to form a stable finger structure, it play an important role in gene expression regulation, cell differentiation, embryonic development,eta.
The function of zinc finger proteins in the animal and human body have been penetratelly researched, for the past few years, through constantly investage, we have find the zinc finger protein also playing important role in plant.participated most kinds of resistance reaction in the plant progress of growth.
Promoter is the originate sequence of genetic transcription,it can discrimination and bind to the RNA polymerase.As an important regulatory element, the research of promoter has became the hot point of molecule biology. To study and research the function of promoter, having the important meaning for us to understand the growth and development growth,investage the fit mechanism of the biology and it habitat,controlling the expression of exogenous in the transgenic plant.The principle consequence of this research is just as follow:
1. Using the nested PCR, obtaining the MsZFN gene’s promoter sequence,about 1500bp, to compare the homology in the blast database,this gene is obtained for the first time, it has 88% similiraty with the Medicago truncatula chromosome 7 clone mth2-36p20, Logging in the Genebank, accession number is FJ161979.1,named MsZPP, To analysis the sequence, found many CAAT-boxes and TATA-boxes, TATA-box can lead the RNA polymeraseⅡto the right transcriptional start site, CAAT-box mainly control the starting frequence of transcription, is an element of regulate transcription starting,having no direct relationship for the specification of the promoter. It also found some potentional cis-acting element, for example, the ABRE ,which is related to the ABA derivation; the MBS,which is related to drought induction; the TGACG-motif, which is related to the jasmonic methylester,and some photoresponse elemant, such as ACE,G-box,GT1-motif, I-box, SpI.
2. constructing the complete sequence expression vector and translating tobacco, obtaining the transgenic plant, PCR detection shows the objective gene has intergrated to the tobacco genome.
3. According to the transcriptional start site which has been predicted, cloning the different sequence which is short of the different length of 5’sequence.Constructed the expression vector, translated tobacco, obtained the transgenic plant, PCR detection shows the objective gene has intergrated to the tobacco genome
启动子(promoter)是RNA聚合酶能够识别并与之结合,从而起始基因转录的序列,作为转录水平上一种重要的调控元件,研究启动子,对我们了解生物的生长发育过程,探讨生物对其生长环境的适应机制以及更好地控制外源基因在转基因植物中的表达等具有重要意义。本研究取得的主要结果如下:
1.应用巢式PCR技术,克隆获得了锌指蛋白基因MsZFN的上游启动子区,大约1500bp左右,在Blast数据库上进行同源性比较,该基因为首次获得,与截形苜蓿7号染色体克隆mth2 - 36p20的全序列有88%的相似性。在GenBank上登录,登录号为FJ161979.1,命名为MsZPP。对获得的序列进行分析,发现了多个CAAT-box和TATA-box,TATA框具有将RNA聚合酶Ⅱ引导到正确的转录起始位点的功能,CAAT框主要控制着转录的起始频率,是调控转录速率的元件,与启动子的特异性无直接的关系。还发现了多个潜在的顺式作用元件,如与ABA诱导相关的顺式作用元件ABA响应元件(ABRE),与干旱诱导有关的MYB结合位点(MBS),与茉莉酸甲酯反应相关的顺式调控元件TGACG-motif,以及多个光响应元ACE,G-box,GT1-motif, I-box, SpI。
2.构建了启动子全长序列的表达载体并运用农杆菌介导的方法转化烟草,获得了转基因植株,PCR检测显示已将目的片段成功整合到烟草的基因组中。
3.根据预测的转录起始位点克隆不同长度的5’端缺失序列,并构建了用于转化的表达载体,运用农杆菌介导的方法转化烟草,获得了转基因植株,PCR检测显示已将目的片段成功整合到烟草的基因组中。
Alfalfa (Medicago sativa L.) is the world's most widely distributed perennial legume forage grass, and also is the most important one of the legumes forage grass. It is not only high yield but also nutrient-rich, so it is called the“king of grass”. Traditional breeding methods is mainly through the hybridization to abtain the new varieties. It required long cycle and heavy workload. Using molecular biology methods is the hot spot of current breeding research, especially applied to the resistance breeding work, it can shorten the breeding period, improve breeding efficiency.This study is mainly about a zinc finger protein gene’promoter. Zinc finger protein is the most rich protein in a class of transcription factors in eukaryotic genome, such proteins binding the Zn2 + to form a stable finger structure, it play an important role in gene expression regulation, cell differentiation, embryonic development,eta.
The function of zinc finger proteins in the animal and human body have been penetratelly researched, for the past few years, through constantly investage, we have find the zinc finger protein also playing important role in plant.participated most kinds of resistance reaction in the plant progress of growth.
Promoter is the originate sequence of genetic transcription,it can discrimination and bind to the RNA polymerase.As an important regulatory element, the research of promoter has became the hot point of molecule biology. To study and research the function of promoter, having the important meaning for us to understand the growth and development growth,investage the fit mechanism of the biology and it habitat,controlling the expression of exogenous in the transgenic plant.The principle consequence of this research is just as follow:
1. Using the nested PCR, obtaining the MsZFN gene’s promoter sequence,about 1500bp, to compare the homology in the blast database,this gene is obtained for the first time, it has 88% similiraty with the Medicago truncatula chromosome 7 clone mth2-36p20, Logging in the Genebank, accession number is FJ161979.1,named MsZPP, To analysis the sequence, found many CAAT-boxes and TATA-boxes, TATA-box can lead the RNA polymeraseⅡto the right transcriptional start site, CAAT-box mainly control the starting frequence of transcription, is an element of regulate transcription starting,having no direct relationship for the specification of the promoter. It also found some potentional cis-acting element, for example, the ABRE ,which is related to the ABA derivation; the MBS,which is related to drought induction; the TGACG-motif, which is related to the jasmonic methylester,and some photoresponse elemant, such as ACE,G-box,GT1-motif, I-box, SpI.
2. constructing the complete sequence expression vector and translating tobacco, obtaining the transgenic plant, PCR detection shows the objective gene has intergrated to the tobacco genome.
3. According to the transcriptional start site which has been predicted, cloning the different sequence which is short of the different length of 5’sequence.Constructed the expression vector, translated tobacco, obtained the transgenic plant, PCR detection shows the objective gene has intergrated to the tobacco genome
引文
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