侧根原基发生相关基因NAC1激素作用特征及RSI-1功能初步研究
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摘要
本文研究了在侧根原基发生与侧根发育过程中起重要作用的拟南芥NACl基因的上游调控区在烟草中的作用特征;同时初步研究了番茄RSI-1基因在烟草整体发育及侧根发生中的作用。初步探讨了将上述两个基因的理论研究成果应用于实际生产的可能性。为获得作物根系发育的可人工调控路径和植物生长物质的生物测定材料作了一些探索性工作。
本研究分两个部分,第一部分研究了NACl上游调控区(启动子)的作用特征。NACl作为一个早期的生长素反应基因,可被生长素诱导并介导生长素促进侧根的发生信号。序列分析结果显示NACl上游调控区除了含有生长素反应元件外还含有多个赤霉素反应元件。该部分研究克隆了其上游调控区,构建了由该调控区驱动GUS、GFP表达的植物表达载体并获得大量的相关转基因烟草。在此基础上分析了转基因烟草中标记基因表达的位置特异性和生长素和赤霉素诱导效应,探讨了该上游调控序列在烟草中的作用特征。结果验证了NACl会在烟草的根尖、侧根原基处特异性表达,发现由NACl上游1kb调控区驱动的GUS还会在烟草侧根原基基部、侧根基部表达。结果在验证了该基因的表达受生长素诱导的基础上,进一步量化了生长素和赤霉素对该基因的表达的诱导效应。结果表明,在0.1mg/L浓度水平,IBA对根中GUS酶量的诱导增加值可达到30%,GA的诱导增加值也能达到20%以上。
研究的第二部分初步探讨了RSI-1基因在植物发育过程中的作用,简要分析了棉花、烟草中存在RSI-1高同源性的基因可能性。Taylor(1994)的研究显示RSI-1可能是控制侧根原基发生的开关基因(Switch point gene),可能在侧根原基发生启动过程中起关键性的作用并决定侧根原基的发生强度。该部分研究分别构建了控制RSI-1超量表达、反义抑制、RNAi干扰的表达载体,对烟草进行了转化并获得大量相应得转基因植株。对所获得的转基因植株及其自交一代的初步表型观察发现RSI-1基因在烟草中超量表达后,地上部分枝能力增强,其T1代在1/2 MS琼脂糖培养基上的长势及根的伸长能力下降。而反义抑制及RNAi干扰的转基因烟草的表型没有发生明显变化。该结果初步说明,RSI-1可能在控制顶端优势过程中发挥作用,同时烟草中不一定存在与RSI-1具有很高同源性的同功能基因,或者RSI-1并不处在是控制某个发育过程的关键性节点上。
A huge root system can lead to high-resistance to stresses and high yield, while it is also a key dissimilatory organ and an extreme root mass can also result in excess energy consumption. As a result, techniques to bring a optimal root mass plays a leading role in crop tillage. The lateral root makes great part of the root system, and the lateral root primordia are the initial. The density of lateral root primordia emergence is usually determined by the express intensity of the key genes controlling the lateral root primordial initiation, while latter is controlled by its promoter. Therefore, characterization of the key genes controlling lateral root primordia initiation and researches of the genes in lateral root development may exert profound influence on manipulate root system development in crop culture.The research is consisted of two parts, and the first part is focus on character of the up-stream region of the NAC1. As an auxin early response gene, it can be induced by IAA and can mediate the auxin signal to promote the lateral root development. Researches show that NAC1 plays an important role in lateral root initiation, and its expression level has a strait effect on the emergence frequence of lateral root primordia. Computer based sequence analysis gives out that besides two auxin response elements local on the up-stream region, there still be several gibberellin response elements. The work here makes of NAC1 up-stream region cloneing, the express vector construction, tobacco transform; the marker gene express manner and the phytohormone induce effect analysis. Data not only verified the preceded result that NAC1 expressed specially in the division zone and in the lateral root primordial, its expression can be induced by auxin, but also show that it can expresses in the base of the lateral root and its expression can also be induced by gibberellin.The second part focused on the RSI-1 function on plant root development. The expression increasing and the enhancement of lateral root primordial emergency share the same manner as induced by auxin. Even more, the increase of RSI-1 transcripts is more early than the enhancement of the lateral root primordia emerge frequence. As a word, its expression and auxin inducing pattern make it like an 'Swithch point gene' in lateral root primordia development. In this part, an over-expression vector, an antisense express vector and an RNAi interference vector were constructed and transferred to tobacco, followed by genotype analysis of the transgenic plants. Result is that the T1 seedlings of the RSI-1 over-expression transformants show a decrease growth competence on 1/2 MS agar media and an increase ramification ability in the field. Evidence show that the RSI-1 protein may plays an role in the apical dominance controlling, and the chance of tobacco has a homologue gene with conserved nucleic sequence region to RSI-1 is slim. Anther possibility is that RSI-1 dose not works on the key point in the lateral root primordia initiation controlling network which is not consist with the result the author descript in the paper.The dissertation gives the works on the auxin and gibberellin induce effect of NAC1 and the phenotype changes in the transgenic tobacco with different vector induced in. The researches are some base works in trying to manipulate the crop root system development. The possibility of the two genes used in crop breedings and in new plant growth substance findings are alsodiscussed.
本研究分两个部分,第一部分研究了NACl上游调控区(启动子)的作用特征。NACl作为一个早期的生长素反应基因,可被生长素诱导并介导生长素促进侧根的发生信号。序列分析结果显示NACl上游调控区除了含有生长素反应元件外还含有多个赤霉素反应元件。该部分研究克隆了其上游调控区,构建了由该调控区驱动GUS、GFP表达的植物表达载体并获得大量的相关转基因烟草。在此基础上分析了转基因烟草中标记基因表达的位置特异性和生长素和赤霉素诱导效应,探讨了该上游调控序列在烟草中的作用特征。结果验证了NACl会在烟草的根尖、侧根原基处特异性表达,发现由NACl上游1kb调控区驱动的GUS还会在烟草侧根原基基部、侧根基部表达。结果在验证了该基因的表达受生长素诱导的基础上,进一步量化了生长素和赤霉素对该基因的表达的诱导效应。结果表明,在0.1mg/L浓度水平,IBA对根中GUS酶量的诱导增加值可达到30%,GA的诱导增加值也能达到20%以上。
研究的第二部分初步探讨了RSI-1基因在植物发育过程中的作用,简要分析了棉花、烟草中存在RSI-1高同源性的基因可能性。Taylor(1994)的研究显示RSI-1可能是控制侧根原基发生的开关基因(Switch point gene),可能在侧根原基发生启动过程中起关键性的作用并决定侧根原基的发生强度。该部分研究分别构建了控制RSI-1超量表达、反义抑制、RNAi干扰的表达载体,对烟草进行了转化并获得大量相应得转基因植株。对所获得的转基因植株及其自交一代的初步表型观察发现RSI-1基因在烟草中超量表达后,地上部分枝能力增强,其T1代在1/2 MS琼脂糖培养基上的长势及根的伸长能力下降。而反义抑制及RNAi干扰的转基因烟草的表型没有发生明显变化。该结果初步说明,RSI-1可能在控制顶端优势过程中发挥作用,同时烟草中不一定存在与RSI-1具有很高同源性的同功能基因,或者RSI-1并不处在是控制某个发育过程的关键性节点上。
A huge root system can lead to high-resistance to stresses and high yield, while it is also a key dissimilatory organ and an extreme root mass can also result in excess energy consumption. As a result, techniques to bring a optimal root mass plays a leading role in crop tillage. The lateral root makes great part of the root system, and the lateral root primordia are the initial. The density of lateral root primordia emergence is usually determined by the express intensity of the key genes controlling the lateral root primordial initiation, while latter is controlled by its promoter. Therefore, characterization of the key genes controlling lateral root primordia initiation and researches of the genes in lateral root development may exert profound influence on manipulate root system development in crop culture.The research is consisted of two parts, and the first part is focus on character of the up-stream region of the NAC1. As an auxin early response gene, it can be induced by IAA and can mediate the auxin signal to promote the lateral root development. Researches show that NAC1 plays an important role in lateral root initiation, and its expression level has a strait effect on the emergence frequence of lateral root primordia. Computer based sequence analysis gives out that besides two auxin response elements local on the up-stream region, there still be several gibberellin response elements. The work here makes of NAC1 up-stream region cloneing, the express vector construction, tobacco transform; the marker gene express manner and the phytohormone induce effect analysis. Data not only verified the preceded result that NAC1 expressed specially in the division zone and in the lateral root primordial, its expression can be induced by auxin, but also show that it can expresses in the base of the lateral root and its expression can also be induced by gibberellin.The second part focused on the RSI-1 function on plant root development. The expression increasing and the enhancement of lateral root primordial emergency share the same manner as induced by auxin. Even more, the increase of RSI-1 transcripts is more early than the enhancement of the lateral root primordia emerge frequence. As a word, its expression and auxin inducing pattern make it like an 'Swithch point gene' in lateral root primordia development. In this part, an over-expression vector, an antisense express vector and an RNAi interference vector were constructed and transferred to tobacco, followed by genotype analysis of the transgenic plants. Result is that the T1 seedlings of the RSI-1 over-expression transformants show a decrease growth competence on 1/2 MS agar media and an increase ramification ability in the field. Evidence show that the RSI-1 protein may plays an role in the apical dominance controlling, and the chance of tobacco has a homologue gene with conserved nucleic sequence region to RSI-1 is slim. Anther possibility is that RSI-1 dose not works on the key point in the lateral root primordia initiation controlling network which is not consist with the result the author descript in the paper.The dissertation gives the works on the auxin and gibberellin induce effect of NAC1 and the phenotype changes in the transgenic tobacco with different vector induced in. The researches are some base works in trying to manipulate the crop root system development. The possibility of the two genes used in crop breedings and in new plant growth substance findings are alsodiscussed.
引文
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