Ri质粒转化的烟草发状根培养及其烟碱生物合成调控的研究
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摘要
本试验研究了发根农杆菌Ri质粒转化烟草的各种影响因素，确定了最佳的转化条件，建立了烟草发根培养系统：对发根与非转化根的形态结构、生长动态进行了研究，并以Ri质粒T-DNA上的rolC基因设计引物，对发根进行了分子检测，利用GUS报告基因检测了外源基因在发根和再生植株上的表达，为发根遗传转化提供了可靠的分子证据；并在悬浮条件下，进行了不同理化因子对烟草毛状根生长和烟碱合成调控的研究。主要结果如下：
1．本研究通过发根农杆菌工程菌LBA1334(含有野生型pRi1855质粒和pIG121HM双元载体质粒，编码NPTⅡ基因和GUS基因)转化烟草沙姆逊，获得了具有卡那霉素抗性烟草发状根，并筛选出良好的单克隆株系，该发状根具有典型的发状根特性(多根毛、多分支、在无激素培养基上快速生长)，通过根尖压片分析，烟草发状根不具备典型的根冠；发状根中烟碱含量1.61％稍高于与天然栽培品种母体根的含量(1.5％左右)，发状根向培养液中释放烟碱，释放量最多时占总烟碱量的81.1％；在无激素的培养基上获得了再生植株。
2．以Ri质粒T-DNA上的rolC基因设计特异引物，对发状根进行PCR扩增，得到了560bp的目的基因片段，证明了Ri质粒在烟草发状根的基因组中得到了整合：本试验还对烟草发状根和再生植株叶片进行了GUS组织化学检测，在烟草发状根中和再生植株叶片中检测到兰色反应，说明了GUS基因在烟草发状根和再生植株中得到表达。以上检测结果表明烟草发状根体系作为载体进行外源基因表达生产目的产物以及进行烟草品种改良的可能性。
3．对烟草发状根诱导的影响因子(诸如发根农杆菌菌株、烟草品种、叶片程度、菌液浓度、稀释倍数、浸染时间、共培养时间等)进行了研究，确定
出最佳的组合条件：以发根农杆菌工程菌株LBA1334和沙姆逊为转化体
系，选取上部幼嫩的组培苗叶片，在稀释5上0倍的对数生长期菌液
（OD60斤0个0．8）中浸泡5分钟，共培养2大，进行脱菌筛选培养，可获
得高的转化效率。
4．悬浮培养中发根的生长和物质合成受多种理化因子的影响（培养基、pH
值、装液量、蔗糖浓度、添加物等）的影响，本实验中，以 1o 液体为
Z 培养基，蔗糖20上0g／L，pH值6刀，装液量为容器体积的2／5，可添加水
解酪蛋白Q）和 IAA门 人在 27”C，11 orpm下暗培养，25天左
右收获发状根，可达最佳效果。
5．烟草发状根培养中，激素对发状根生长和烟碱合成有较大影响。赤霉素
（GA3）可显著增加发状根的生长量，但抑制烟碱的生成；u跺乙酸uAA）
在合适浓度门mg／L）即能增加发状根的生长又能促进烟碱的合成；＃乙
酸（NAA）对发状根生长量和烟碱的生成无明显促进作用。
6．通过对烟草发状根培养液pH值的研究，表明烟草发状根在培养的最初8
大内，培养液pH值变化到5刀左右，一直维持到悬浮培养后期；收获时
培养液pH值与发状根生物总量呈显著的正相关。
Factors affecting the transformation of tobacco by Ri plasmid of Agrobacterium rhizogene were studied and the optimum transformation condition was defined. The structure of the hairy root tip and the dynamic characteristics of growth of a selected hairy root line were studied. The transformation of hairy root was verified by PCR test and GUS histochemistry test. The physical and chemical factors that affect the growth and nicotine biosynthesis of hairy root were investigated. The main results were as follows:
1. Hairy roots were induced from tobacco leaves with Agrobacterium rhizogenes LBA1334 harboring agropin type PRil855 and binary vector PIG121HM carrying a kanamycin resistant gene nptll and GUS gene and the good hairy root clones were selected. The root with many root hairs and branches grew vigorously toward all the direction on MS medium without plant hormone and on kanamyain containing medium (km:30mg/L). The structure of the hairy root tip is different from that of ordinary root. Witch was not typical root cap maybe the main cause of losing the geotropism of hairy root. Plantlet regenerated from hairy root when cultured on Ms medium without plant hormone. The nicotine content of the hairy root was little higher than that in the natural roots. The culture system of hairy root could release nicotine into the medium and the amount of nicotine was 81.1% of the total content of nicotine produced by the culture system.
2. A 560 bp fragment was amplified from the genomic DNA of hairy root by PCR using primes of rolC which was one of the four genes related to hairy root induction located on TL-DNA of Ri plasmid. Blue dots were discovered on the hairy root and the leaves of planted regenerated from hairy root after dipped into X-gluc solution at 37℃ for one night. These results proved tobacco hairy root could be an efficient culture system to accumulate valuable substance and an ideal pathway for plant breeding through exogenous genes expressing.
3. Factors affecting the transformation of tobacco by Ri plasmid of Agrobacterium rhizogene were studied. Tobacco varieties. Agrobacterium rhizogene strains and basal medium influenced the efficiency of hairy root formation strikingly. While the PH of medium and acetosyringone had little effects. MS medium and strain LBA1334 give the best result on tobacco transformation. Sha Mu Xun was more sensitive to Agrobacterium rhizogene than K326 and NC89. Leaf discs dipped into bacterium solution diluted 5 times at exponential phase for 5 minutes, and pre-cultured on MS medium for two days was very useful preparation for transformation.
48
4. The growth of the hairy root and nicotine biosynthesis were influenced by various physical and chemical factors. It was found that the optimum pH value of medium was 6.0. Fast growth of the hairy root and maximal production of nicotine was observed in l/2medium and in the presence of 2-3% sucrose. The best volume of liquid medium for the growth and nicotine synthesis of the hairy root was 2/5 of the volume of container at 110rpm. IAA(lmg/L)and LH(2g/L)also enhanced the weight and nicotine content of the hairy root.
5. Phytohormones affected obviously the growth and nicotine content of the hairy root. GA3 improved remarkably the growth speed of the hairy root but the nicotine synthesis was inhibited. IAA could raise the weight and the nicotine content of the hairy root when the concentration of IAA was 1mg/L. The growth and nicotine production in hairy root cultures weren't greatly promoted by addition of NAA.
6. The medium pH value was studied during the culture process. The results show that the medium pH value was 5.0 at the beginning of cultivation, and the pH could be kept till the stationary stage. The medium pH increased linearly relative to the biomass accumulation of the hairy root.
1．本研究通过发根农杆菌工程菌LBA1334(含有野生型pRi1855质粒和pIG121HM双元载体质粒，编码NPTⅡ基因和GUS基因)转化烟草沙姆逊，获得了具有卡那霉素抗性烟草发状根，并筛选出良好的单克隆株系，该发状根具有典型的发状根特性(多根毛、多分支、在无激素培养基上快速生长)，通过根尖压片分析，烟草发状根不具备典型的根冠；发状根中烟碱含量1.61％稍高于与天然栽培品种母体根的含量(1.5％左右)，发状根向培养液中释放烟碱，释放量最多时占总烟碱量的81.1％；在无激素的培养基上获得了再生植株。
2．以Ri质粒T-DNA上的rolC基因设计特异引物，对发状根进行PCR扩增，得到了560bp的目的基因片段，证明了Ri质粒在烟草发状根的基因组中得到了整合：本试验还对烟草发状根和再生植株叶片进行了GUS组织化学检测，在烟草发状根中和再生植株叶片中检测到兰色反应，说明了GUS基因在烟草发状根和再生植株中得到表达。以上检测结果表明烟草发状根体系作为载体进行外源基因表达生产目的产物以及进行烟草品种改良的可能性。
3．对烟草发状根诱导的影响因子(诸如发根农杆菌菌株、烟草品种、叶片程度、菌液浓度、稀释倍数、浸染时间、共培养时间等)进行了研究，确定
出最佳的组合条件：以发根农杆菌工程菌株LBA1334和沙姆逊为转化体
系，选取上部幼嫩的组培苗叶片，在稀释5上0倍的对数生长期菌液
（OD60斤0个0．8）中浸泡5分钟，共培养2大，进行脱菌筛选培养，可获
得高的转化效率。
4．悬浮培养中发根的生长和物质合成受多种理化因子的影响（培养基、pH
值、装液量、蔗糖浓度、添加物等）的影响，本实验中，以 1o 液体为
Z 培养基，蔗糖20上0g／L，pH值6刀，装液量为容器体积的2／5，可添加水
解酪蛋白Q）和 IAA门 人在 27”C，11 orpm下暗培养，25天左
右收获发状根，可达最佳效果。
5．烟草发状根培养中，激素对发状根生长和烟碱合成有较大影响。赤霉素
（GA3）可显著增加发状根的生长量，但抑制烟碱的生成；u跺乙酸uAA）
在合适浓度门mg／L）即能增加发状根的生长又能促进烟碱的合成；＃乙
酸（NAA）对发状根生长量和烟碱的生成无明显促进作用。
6．通过对烟草发状根培养液pH值的研究，表明烟草发状根在培养的最初8
大内，培养液pH值变化到5刀左右，一直维持到悬浮培养后期；收获时
培养液pH值与发状根生物总量呈显著的正相关。
Factors affecting the transformation of tobacco by Ri plasmid of Agrobacterium rhizogene were studied and the optimum transformation condition was defined. The structure of the hairy root tip and the dynamic characteristics of growth of a selected hairy root line were studied. The transformation of hairy root was verified by PCR test and GUS histochemistry test. The physical and chemical factors that affect the growth and nicotine biosynthesis of hairy root were investigated. The main results were as follows:
1. Hairy roots were induced from tobacco leaves with Agrobacterium rhizogenes LBA1334 harboring agropin type PRil855 and binary vector PIG121HM carrying a kanamycin resistant gene nptll and GUS gene and the good hairy root clones were selected. The root with many root hairs and branches grew vigorously toward all the direction on MS medium without plant hormone and on kanamyain containing medium (km:30mg/L). The structure of the hairy root tip is different from that of ordinary root. Witch was not typical root cap maybe the main cause of losing the geotropism of hairy root. Plantlet regenerated from hairy root when cultured on Ms medium without plant hormone. The nicotine content of the hairy root was little higher than that in the natural roots. The culture system of hairy root could release nicotine into the medium and the amount of nicotine was 81.1% of the total content of nicotine produced by the culture system.
2. A 560 bp fragment was amplified from the genomic DNA of hairy root by PCR using primes of rolC which was one of the four genes related to hairy root induction located on TL-DNA of Ri plasmid. Blue dots were discovered on the hairy root and the leaves of planted regenerated from hairy root after dipped into X-gluc solution at 37℃ for one night. These results proved tobacco hairy root could be an efficient culture system to accumulate valuable substance and an ideal pathway for plant breeding through exogenous genes expressing.
3. Factors affecting the transformation of tobacco by Ri plasmid of Agrobacterium rhizogene were studied. Tobacco varieties. Agrobacterium rhizogene strains and basal medium influenced the efficiency of hairy root formation strikingly. While the PH of medium and acetosyringone had little effects. MS medium and strain LBA1334 give the best result on tobacco transformation. Sha Mu Xun was more sensitive to Agrobacterium rhizogene than K326 and NC89. Leaf discs dipped into bacterium solution diluted 5 times at exponential phase for 5 minutes, and pre-cultured on MS medium for two days was very useful preparation for transformation.
48
4. The growth of the hairy root and nicotine biosynthesis were influenced by various physical and chemical factors. It was found that the optimum pH value of medium was 6.0. Fast growth of the hairy root and maximal production of nicotine was observed in l/2medium and in the presence of 2-3% sucrose. The best volume of liquid medium for the growth and nicotine synthesis of the hairy root was 2/5 of the volume of container at 110rpm. IAA(lmg/L)and LH(2g/L)also enhanced the weight and nicotine content of the hairy root.
5. Phytohormones affected obviously the growth and nicotine content of the hairy root. GA3 improved remarkably the growth speed of the hairy root but the nicotine synthesis was inhibited. IAA could raise the weight and the nicotine content of the hairy root when the concentration of IAA was 1mg/L. The growth and nicotine production in hairy root cultures weren't greatly promoted by addition of NAA.
6. The medium pH value was studied during the culture process. The results show that the medium pH value was 5.0 at the beginning of cultivation, and the pH could be kept till the stationary stage. The medium pH increased linearly relative to the biomass accumulation of the hairy root.
引文
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