盐芥激活标签突变体库的构建及盐芥转座子标签突变体的筛选
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摘要
植物对盐胁迫的适应是一个非常复杂的过程,如何提高作物的耐盐性仍然面临着极大的挑战。对植物在受到盐胁迫时所作出的反应进行分析,寻找植物基因组中与耐盐相关的基因,进一步研究这些基因的反应机制,不仅对于揭示植物耐逆的机理具有重要的理论意义,而且对于耐盐作物的培育具有重要的实践意义。
盐芥是拟南芥的一种近缘植物,与拟南芥一样,盐芥也具有很多可以被做为分子生物学研究模式系统的优点:比如说短的生活史、小的基因组、易于被转化以及丰富的种子等等。盐芥和拟南芥在cDNA和氨基酸水平上分别有95%和90%的同源性,这样在开展研究时,我们就可以方便地将拟南芥的很多信息(比如说基因、蛋白质数据库以及突变体系等)用于盐芥耐盐性的分子生物学研究。但是,与拟南芥不同的是盐芥非常抗盐,短时间内能耐受高达500mMNaCl的冲击,并且在受到盐胁迫时既不产生盐腺也没有发生复杂的形态上的变化,这些结果说明盐芥的耐盐性很大程度上源于它自身基本的生理和生化机制。目前,盐芥已经成为一种非常有研究价值的耐盐模式系统,人们已经先后开展了对盐芥生理生化、基因表达的研究,同时还建立了各种cDNA文库和突变体库。
盐芥基因组的测序即将完成,功能基因组学研究将会成为我们以后研究工作的重点,而通过各种不同的方法建立大规模的突变体库是目前植物功能基因组学研究的最直接也是最有效的方法。从目前来看,在进行突变体建库时用到的方法主要有以下几种:自发突变、理化诱变和插入突变,其中插入突变又可以分为T-DNA插入突变和转座子标签两种方法。
本实验以植物耐盐模式系统盐芥为研究材料,通过农杆菌介导的花浸染法将激活标记载体pSKI015、转座子标签Ds launch pad的T-DNA载体分别导入盐芥基因组,试图建立盐芥插入突变体库。对初步获得的抗性植株,进行了分子检测以及侧翼序列的克隆与分析。本实验仅对盐芥插入突变体库的建立进行了初步的工作,大规模的突变体库正在构建之中。
本论文的主要目的是建立耐盐模式植物盐芥的激活标签突变体库,同时还对转座子标签法得到盐芥突变体的方法进行了初步的研究。主要结果如下:
1.盐芥激活标签突变体库的建立
本实验是通过农杆菌介导的花浸染法将激活标记载体pSKI015导入盐芥基因组,试图建立一定规模的盐芥激活标记突变体库。实验中得到的结果主要有以下几个方面,
(1)利用激活标记载体pSKI015对盐芥进行遗传转化,共获得转化T0代种子800g左右,通过除草剂(Basta)筛选,共获得抗性苗约853株。
(2)对得到的抗性苗进行检测,是通过对Bar基因的PCR检测来进行的。检测结果发现在853株抗性苗中,共有685株阳性苗,阳性率在80%左右。
(3)采用TAIL-PCR的方法对得到的阳性苗进行T-DNA插入位点侧翼序列的扩增,并进行测序,得到侧翼序列36条,并进行了相关的序列分析。
2.盐芥转座子标签突变体的筛选
通过农杆菌介导的花浸染法将转座子标签的Ds载体导入盐芥的基因组中,然后进行筛选,得到突变体。得到的结果主要有:
(1)利用Ds载体对盐芥进行遗传转化,通过除草剂(Basta)筛选,共获得抗性苗约87株。
(2)对获得的抗性苗进行PCR检测,是通过对Bar基因的PCR检测来进行的。结果发现在87株抗性苗中共有76株阳性苗。
The mechanism of plants adaptation to salt stress is very complex, so improving the salt tolerance of crops is still a great challenge. Therefore, analyzing the mechanism of plants responses to salt stress, exploring genes related to salt tolerance not only have important theoretical significance, but also have important practical significance for cultivation of salt-tolerant crops.
Thellungiella halophila (Salt cress) is a close relative of Arabidopsis thaliana, and has good genetic features such as a short life cycle, a small size of genome, an efficient transformation and high seed numbers, etc. Thellungiella halophila and Arabidopsis thaliana share 95% and 90% identities on cDNA and amino acid sequences respectively, so it is convenient to transfer informations of Arabidopsis (gene database, protein database and mutant lines) to molecular analysis on salt tolerance of salt cress. But, in sharp contrast with Arabidopsis, Thellungiella is able to withstand dramatic salinity shock up to 500 mM NaCl. This plant does not produce salt glands or other complex morphological alterations either before or after salt adaption. Up to now, Thellungiella has become a valuable model for the study of salt tolerance. And analysis on salt cress physiology, biochemistry and gene expression, and the construction of different cDNA libraries and mutant libraries have been made.
Since Thellungiella sequencing is to be completed, the main task of this work will be functional genomic study, and the most direct and efficient way of functional genome study is to construct large-scale mutant library by various kinds of methods. From the current view, the mutant library construction methods used are mainly the following: spontaneous mutation, physical and chemical mutagenesis and insertional mutagenesis, in which insertion mutations can be divided into T-DNA insertional mutagenesis and transposon tagging methods.
In this experiment, activation tagging vector pSKI015 and Ds launch pad T-DNA vector were respectively introduced into Thellungiella by Agrobacterium tumefaciens-mediated flora dip transformation with an intention to construct an insertional mutant library. Molecular analysis and cloning of the flanking genomic sequences have been conducted to the obtained resistant plants. Summarily, in this experiment, only primary research was made on construction of Thellungiella insertional mutant library and the library in large scale is being constructed now.
The main objectives of the dissertation were to construct an activation tagging mutant library of Thellungiella and the transposon tagging method to obtain Thellungiella mutants had also been explored. The main results are as following:
1.The construction of activation tagging mutant library of Thellungiella
In this experiment, activation tagging vector pSKI015 was introduced into Thellungiella by Agrobacterium tumefaciens-mediated flora dip transformation with an intention to construct an activation-tagged mutant library. The main results are as following:
(1) Activation tagging vector pSKI015 was introduced into the salt tolerance model plant Thellungiella halophila by Agrobacterium tumefaciens-mediated flora dipping method, we have obtained nearly 800g of T0 transgenic seeds, and 853 lines of Basta-resistant Thellungiella seedlings by Basta screening.
(2) The basta-resistant Thellungiella plants were further identified by PCR detection of Bar gene, and the results showed that 685 of 853 resistant seedlings were positive, the positive rate reached 80%.
(3) TAIL-PCR method were used to obtain the flanking genomic sequences of T-DNA insertion site from the 685 Bar-resistant Thellungiella seedlings, about 36 flanking sequences have been obtained, and BLAST analysis of these sequences have been done.
2. The selection of Thellungiella transposon-tagged mutants Transposon tagged vector Ds was introduced into Thellungiella halophila by Agrobacterium tumefaciens-mediated flora dipping transformation.Then we selected transgenic T0 plants by Basta. And we obtained resistant Thellungiella seedlings. The main results were as below:
(1) Transposon tagged vector Ds was introduced into Thellungiella halophila, we obtained transgenic T0 seeds, selected by Basta, about 87 Basta-resistant Thellungiella seedlings were obtained.
(2) The results of PCR detection of Bar gene in resistant plants found that,76 lines of 87 resistant seedlings were positive.
盐芥是拟南芥的一种近缘植物,与拟南芥一样,盐芥也具有很多可以被做为分子生物学研究模式系统的优点:比如说短的生活史、小的基因组、易于被转化以及丰富的种子等等。盐芥和拟南芥在cDNA和氨基酸水平上分别有95%和90%的同源性,这样在开展研究时,我们就可以方便地将拟南芥的很多信息(比如说基因、蛋白质数据库以及突变体系等)用于盐芥耐盐性的分子生物学研究。但是,与拟南芥不同的是盐芥非常抗盐,短时间内能耐受高达500mMNaCl的冲击,并且在受到盐胁迫时既不产生盐腺也没有发生复杂的形态上的变化,这些结果说明盐芥的耐盐性很大程度上源于它自身基本的生理和生化机制。目前,盐芥已经成为一种非常有研究价值的耐盐模式系统,人们已经先后开展了对盐芥生理生化、基因表达的研究,同时还建立了各种cDNA文库和突变体库。
盐芥基因组的测序即将完成,功能基因组学研究将会成为我们以后研究工作的重点,而通过各种不同的方法建立大规模的突变体库是目前植物功能基因组学研究的最直接也是最有效的方法。从目前来看,在进行突变体建库时用到的方法主要有以下几种:自发突变、理化诱变和插入突变,其中插入突变又可以分为T-DNA插入突变和转座子标签两种方法。
本实验以植物耐盐模式系统盐芥为研究材料,通过农杆菌介导的花浸染法将激活标记载体pSKI015、转座子标签Ds launch pad的T-DNA载体分别导入盐芥基因组,试图建立盐芥插入突变体库。对初步获得的抗性植株,进行了分子检测以及侧翼序列的克隆与分析。本实验仅对盐芥插入突变体库的建立进行了初步的工作,大规模的突变体库正在构建之中。
本论文的主要目的是建立耐盐模式植物盐芥的激活标签突变体库,同时还对转座子标签法得到盐芥突变体的方法进行了初步的研究。主要结果如下:
1.盐芥激活标签突变体库的建立
本实验是通过农杆菌介导的花浸染法将激活标记载体pSKI015导入盐芥基因组,试图建立一定规模的盐芥激活标记突变体库。实验中得到的结果主要有以下几个方面,
(1)利用激活标记载体pSKI015对盐芥进行遗传转化,共获得转化T0代种子800g左右,通过除草剂(Basta)筛选,共获得抗性苗约853株。
(2)对得到的抗性苗进行检测,是通过对Bar基因的PCR检测来进行的。检测结果发现在853株抗性苗中,共有685株阳性苗,阳性率在80%左右。
(3)采用TAIL-PCR的方法对得到的阳性苗进行T-DNA插入位点侧翼序列的扩增,并进行测序,得到侧翼序列36条,并进行了相关的序列分析。
2.盐芥转座子标签突变体的筛选
通过农杆菌介导的花浸染法将转座子标签的Ds载体导入盐芥的基因组中,然后进行筛选,得到突变体。得到的结果主要有:
(1)利用Ds载体对盐芥进行遗传转化,通过除草剂(Basta)筛选,共获得抗性苗约87株。
(2)对获得的抗性苗进行PCR检测,是通过对Bar基因的PCR检测来进行的。结果发现在87株抗性苗中共有76株阳性苗。
The mechanism of plants adaptation to salt stress is very complex, so improving the salt tolerance of crops is still a great challenge. Therefore, analyzing the mechanism of plants responses to salt stress, exploring genes related to salt tolerance not only have important theoretical significance, but also have important practical significance for cultivation of salt-tolerant crops.
Thellungiella halophila (Salt cress) is a close relative of Arabidopsis thaliana, and has good genetic features such as a short life cycle, a small size of genome, an efficient transformation and high seed numbers, etc. Thellungiella halophila and Arabidopsis thaliana share 95% and 90% identities on cDNA and amino acid sequences respectively, so it is convenient to transfer informations of Arabidopsis (gene database, protein database and mutant lines) to molecular analysis on salt tolerance of salt cress. But, in sharp contrast with Arabidopsis, Thellungiella is able to withstand dramatic salinity shock up to 500 mM NaCl. This plant does not produce salt glands or other complex morphological alterations either before or after salt adaption. Up to now, Thellungiella has become a valuable model for the study of salt tolerance. And analysis on salt cress physiology, biochemistry and gene expression, and the construction of different cDNA libraries and mutant libraries have been made.
Since Thellungiella sequencing is to be completed, the main task of this work will be functional genomic study, and the most direct and efficient way of functional genome study is to construct large-scale mutant library by various kinds of methods. From the current view, the mutant library construction methods used are mainly the following: spontaneous mutation, physical and chemical mutagenesis and insertional mutagenesis, in which insertion mutations can be divided into T-DNA insertional mutagenesis and transposon tagging methods.
In this experiment, activation tagging vector pSKI015 and Ds launch pad T-DNA vector were respectively introduced into Thellungiella by Agrobacterium tumefaciens-mediated flora dip transformation with an intention to construct an insertional mutant library. Molecular analysis and cloning of the flanking genomic sequences have been conducted to the obtained resistant plants. Summarily, in this experiment, only primary research was made on construction of Thellungiella insertional mutant library and the library in large scale is being constructed now.
The main objectives of the dissertation were to construct an activation tagging mutant library of Thellungiella and the transposon tagging method to obtain Thellungiella mutants had also been explored. The main results are as following:
1.The construction of activation tagging mutant library of Thellungiella
In this experiment, activation tagging vector pSKI015 was introduced into Thellungiella by Agrobacterium tumefaciens-mediated flora dip transformation with an intention to construct an activation-tagged mutant library. The main results are as following:
(1) Activation tagging vector pSKI015 was introduced into the salt tolerance model plant Thellungiella halophila by Agrobacterium tumefaciens-mediated flora dipping method, we have obtained nearly 800g of T0 transgenic seeds, and 853 lines of Basta-resistant Thellungiella seedlings by Basta screening.
(2) The basta-resistant Thellungiella plants were further identified by PCR detection of Bar gene, and the results showed that 685 of 853 resistant seedlings were positive, the positive rate reached 80%.
(3) TAIL-PCR method were used to obtain the flanking genomic sequences of T-DNA insertion site from the 685 Bar-resistant Thellungiella seedlings, about 36 flanking sequences have been obtained, and BLAST analysis of these sequences have been done.
2. The selection of Thellungiella transposon-tagged mutants Transposon tagged vector Ds was introduced into Thellungiella halophila by Agrobacterium tumefaciens-mediated flora dipping transformation.Then we selected transgenic T0 plants by Basta. And we obtained resistant Thellungiella seedlings. The main results were as below:
(1) Transposon tagged vector Ds was introduced into Thellungiella halophila, we obtained transgenic T0 seeds, selected by Basta, about 87 Basta-resistant Thellungiella seedlings were obtained.
(2) The results of PCR detection of Bar gene in resistant plants found that,76 lines of 87 resistant seedlings were positive.
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