人工小RNA沉默拟南芥植物型PEPC基因的研究
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摘要
磷酸烯醇式丙酮酸羧化酶(PEPCase)是一种具有多种生理功能的细胞质酶,广泛分布于高等植物、藻类和多数细菌中。在C4和CAM植物中,PEPC负责光合作用中无机碳的初始同化；C3植物的PEPC在促进TCA循环中间代谢物的回补、协调碳和氮的代谢等方面有重要作用,被认为是控制C3植物蛋白质与油脂含量比的一个关键酶。同时,PEPC可能与植物对逆境胁迫的响应有密切关系。
拟南芥(Arabidopsis thaliana)与油菜(Brassica napus L.)同属十字花科芸薹属,亲缘关系非常近,在编码区,基因平均相似性达85%左右,因此,拟南芥相关的功能基因研究对油菜育种具有重要参考价值。
为探索植物型PEPC基因对模式植物拟南芥脂肪酸含量和组分,以及抗逆性等方面的影响,本试验构建了同时沉默拟南芥植物型PEPC基因家族(Atppcl、Atppc2和Atppc3)的人工小RNA植物表达载体,用花序浸染法转化拟南芥,成功获得转化植株,并对转化植株进行了相关的分子鉴定和功能分析。
主要研究结果如下：
1.登录人工小RNA设计网站WMD3,提交Atppcl、Atppc2和Atppc3的cDNA序列,设计相应扩增引物,以pRS300质粒为模板(含拟南芥内源miR319a前体骨架),采用重叠PCR的方法克隆了人工小RNA的前体片段,连入双元表达载体pFGC5941,构建了沉默Atppc1、Atppc2和Atppc3的人工小RNA表达载体pFGC-amiAtppc123,利用花序浸染法转化野生型拟南芥。
2.经过除草剂初步筛选,T1代拟南芥幼苗大部分黄化枯死,少数植株正常生长,具有除草剂抗性。提取除草剂抗性苗的叶片DNA进行PCR检测,获得18株T1代转化植株。人工小RNA的半定量RT-PCR的分析表明,在转化植株中,人工小RNA进行了超量表达。对Atppc1、Atppc2和Atppc3的半定量RT-PCR分析显示：与对照相比较,转化植株Atppc1的表达量显著下调,Atppc2和Atppc3的表达量无明显变化。
3.在NaCl胁迫下,对照和转化植株中,Atppcl和Atppc3表现的更为敏感,在盐处理期间,它们的表达水平呈现不同变化趋势,而Atppc2的表达则相对稳定。T2代转化株系种子含油量与对照相比没有明显差异,而脂肪酸组分的测定结果显示,转化株系种子低碳饱和脂肪酸的比例稍高。
As one of cytoplasmic enzymes, Phosphoenolpyruvate carboxylase(PEPCase) carries out a variety of physiological functions in vivo. It is widely distributed in higher plants, algae and most bacteria. In C4 and CAM plants, phosphoenolpyruvate carboxylase plays an important role in the initial CO2 fixation in photosynthesis. In C3 plants, PEPCase is proposed to be a key enzyme to control the ratio of protein/lipid due to its key contribution in compensating intermediate metabolites in tricarboxylic acid cycle, and balancing carbon/nitrogen metabolism. Meanwhile, PEPC may be closely related to the environmental stress on the plants.
Arabidopsis thaliana and oilseed rape (Brassica napus L.) belong to Cruciferae, Brassica. They have close genetic relationship, existing on average about 85% similarity in the coding region of the genomes. Therefore, studies on A. thaliana provide important reference for rapeseed breeding.
To investigate the effects of plant-type PEPCs on modifying lipid content, fatty acid composition and the response to environmental stress in A. thaliana, the plant-type PEPCs genes of A. thaliana were designed to be silenced by the artificial miRNA (amiRNA).We constructed the plant expression vector and transformed it into wild type A. thaliana (col-0) by the floral dip method. In this way, we successfully accquired transgenic plants. The related work of molecular identification and genetic analysis was carried out.
Main results of this study are followed:
1. We input the cDNA sequence of Atppc1, Atppc2 and Atppc3 into the website of WMD3 to design the artifical miRNAs online. Then, we synthesized the related primers to clone the precursor fragment of amiRNA by overlapping PCR, with the pRS300 plasmid as incipient template, which contains the precursor backbone of miR319a, one of the endogenous miRNAs in A. thaliana. Subsequently, we cloned the precursor fragment of amiRNA into the binary expression vector of pFGC5941 to obtain the plant transformation vector, named pFGC-amiAtppc123, and then transformed it into A. thaliana by the floral dip method.
2. With the herbicide basta spraying, most To generation seedlings withered, but still some of them grew normally. We chose 18 normally growing plants for molecular assessment. All of them were successful transformants, which were confirmed by PCR, with the leaves genomic DNA as templates. The amiRNA transcription levels were assessed by semi-quantitative RT-PCR. The results showed that amiRNA transcription level was drastically increased in transgenic plants compared with non-transgenic plants. The results of semi-quantitative RT-PCR showed that the intact mRNA level of Atppcl was drastically suppressed in transgenic plants, compared with non-transgenic plants. However, the expression of Atppc2 and Atppc3 were not significantly changed.
3. Under the stress of NaCl, Atppcl and Atppc3 showed different transcription profile between negative control and transgenic plants. Nevertheless, transcription of Atppc2 was relatively stable. Assessment on the T2 generation transgenic seeds showed oil content of transgenic plants had no significant improvement, compared with non-transgenic plants, but fatty acids composition was slightly adjusted with higher low-carbon saturated fatty acids content.
拟南芥(Arabidopsis thaliana)与油菜(Brassica napus L.)同属十字花科芸薹属,亲缘关系非常近,在编码区,基因平均相似性达85%左右,因此,拟南芥相关的功能基因研究对油菜育种具有重要参考价值。
为探索植物型PEPC基因对模式植物拟南芥脂肪酸含量和组分,以及抗逆性等方面的影响,本试验构建了同时沉默拟南芥植物型PEPC基因家族(Atppcl、Atppc2和Atppc3)的人工小RNA植物表达载体,用花序浸染法转化拟南芥,成功获得转化植株,并对转化植株进行了相关的分子鉴定和功能分析。
主要研究结果如下：
1.登录人工小RNA设计网站WMD3,提交Atppcl、Atppc2和Atppc3的cDNA序列,设计相应扩增引物,以pRS300质粒为模板(含拟南芥内源miR319a前体骨架),采用重叠PCR的方法克隆了人工小RNA的前体片段,连入双元表达载体pFGC5941,构建了沉默Atppc1、Atppc2和Atppc3的人工小RNA表达载体pFGC-amiAtppc123,利用花序浸染法转化野生型拟南芥。
2.经过除草剂初步筛选,T1代拟南芥幼苗大部分黄化枯死,少数植株正常生长,具有除草剂抗性。提取除草剂抗性苗的叶片DNA进行PCR检测,获得18株T1代转化植株。人工小RNA的半定量RT-PCR的分析表明,在转化植株中,人工小RNA进行了超量表达。对Atppc1、Atppc2和Atppc3的半定量RT-PCR分析显示：与对照相比较,转化植株Atppc1的表达量显著下调,Atppc2和Atppc3的表达量无明显变化。
3.在NaCl胁迫下,对照和转化植株中,Atppcl和Atppc3表现的更为敏感,在盐处理期间,它们的表达水平呈现不同变化趋势,而Atppc2的表达则相对稳定。T2代转化株系种子含油量与对照相比没有明显差异,而脂肪酸组分的测定结果显示,转化株系种子低碳饱和脂肪酸的比例稍高。
As one of cytoplasmic enzymes, Phosphoenolpyruvate carboxylase(PEPCase) carries out a variety of physiological functions in vivo. It is widely distributed in higher plants, algae and most bacteria. In C4 and CAM plants, phosphoenolpyruvate carboxylase plays an important role in the initial CO2 fixation in photosynthesis. In C3 plants, PEPCase is proposed to be a key enzyme to control the ratio of protein/lipid due to its key contribution in compensating intermediate metabolites in tricarboxylic acid cycle, and balancing carbon/nitrogen metabolism. Meanwhile, PEPC may be closely related to the environmental stress on the plants.
Arabidopsis thaliana and oilseed rape (Brassica napus L.) belong to Cruciferae, Brassica. They have close genetic relationship, existing on average about 85% similarity in the coding region of the genomes. Therefore, studies on A. thaliana provide important reference for rapeseed breeding.
To investigate the effects of plant-type PEPCs on modifying lipid content, fatty acid composition and the response to environmental stress in A. thaliana, the plant-type PEPCs genes of A. thaliana were designed to be silenced by the artificial miRNA (amiRNA).We constructed the plant expression vector and transformed it into wild type A. thaliana (col-0) by the floral dip method. In this way, we successfully accquired transgenic plants. The related work of molecular identification and genetic analysis was carried out.
Main results of this study are followed:
1. We input the cDNA sequence of Atppc1, Atppc2 and Atppc3 into the website of WMD3 to design the artifical miRNAs online. Then, we synthesized the related primers to clone the precursor fragment of amiRNA by overlapping PCR, with the pRS300 plasmid as incipient template, which contains the precursor backbone of miR319a, one of the endogenous miRNAs in A. thaliana. Subsequently, we cloned the precursor fragment of amiRNA into the binary expression vector of pFGC5941 to obtain the plant transformation vector, named pFGC-amiAtppc123, and then transformed it into A. thaliana by the floral dip method.
2. With the herbicide basta spraying, most To generation seedlings withered, but still some of them grew normally. We chose 18 normally growing plants for molecular assessment. All of them were successful transformants, which were confirmed by PCR, with the leaves genomic DNA as templates. The amiRNA transcription levels were assessed by semi-quantitative RT-PCR. The results showed that amiRNA transcription level was drastically increased in transgenic plants compared with non-transgenic plants. The results of semi-quantitative RT-PCR showed that the intact mRNA level of Atppcl was drastically suppressed in transgenic plants, compared with non-transgenic plants. However, the expression of Atppc2 and Atppc3 were not significantly changed.
3. Under the stress of NaCl, Atppcl and Atppc3 showed different transcription profile between negative control and transgenic plants. Nevertheless, transcription of Atppc2 was relatively stable. Assessment on the T2 generation transgenic seeds showed oil content of transgenic plants had no significant improvement, compared with non-transgenic plants, but fatty acids composition was slightly adjusted with higher low-carbon saturated fatty acids content.
引文
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