【作者】 何新华；

【导师】 陈力耕；

【作者基本信息】 浙江大学 ， 果树学， 2004， 博士

【摘要】 杨梅（Myrica rubra Sieb．et Zucc）系我国特产水果，也是我国南方的一种先锋树种。因其能与弗兰克氏（Frankia）放线菌共生在根部形成固氮根瘤，能将大气中分子态的氮还原成氨供植物吸收利用，能在瘠薄土壤和较恶劣的环境中生长，是改良土壤、治理水土流失、改善生态环境的优良经济生态林果，经济效益显著且经济年限较长，已被国家林业局列为生态公益林备选树种之一。近年来，随着产业结构的调整和山区综合开发，杨梅种植面积迅速扩大，增长势头非常强劲，已成为山区开发的重要经济林树种。 氮素固定是我们这个星球上维持生产力的一个重要生态反应，生物固氮是解决人类目前面临人口、粮食、能源和环境这一总体问题的一个重要方面。如何充分利用杨梅结瘤固氮特性，充分发挥其经济效益和生态环境效益，在农业可持续发展战略中显得尤为重要。 目前有关杨梅共生固氮的研究报道较少，为了更好地开发和利用杨梅共生固氮特性，本论文围绕杨梅根瘤及其共生弗兰克氏菌进行了深入的研究，主要实验结果如下： 1．杨梅根瘤Frankia菌的分离鉴定。采集不同生态环境不同杨梅品种根瘤，采用根瘤切片法和根瘤匀浆悬液法在不同的分离培养基上分离培养。对获得的放线菌菌落进一步纯化，纯化后的菌落进行显微镜检查、固氮酶活性测定、革兰氏染色鉴定，对部分菌株进行回接试验和PCR分子检测，获得了一些弗兰克氏菌株，对其中的3个分离株进行回接试验，能使杨梅幼苗形成根瘤。Q⁺⁺和J-A培养基是分离杨梅根瘤弗兰克氏菌较好的培养基，且Q⁺⁺培养基优于J-A培养基。分离株在Q⁺⁺培养基上表现不同的颜色，呈革兰氏阳性。菌丝分枝，有隔，直径为0.6～1.1 μm；在菌丝末端可以看到圆形顶囊结构，有隔或无隔，直径为1.7～2.2 μm；孢子囊直径15～30μm，梨形或椭圆形；孢子圆形，直径1.4～1.7μm。这些分离株在无氮BAP培养基上表现出乙炔还原活性。 2．Frankia菌培养特性的研究。通过形态观察和Bradford改良法测定菌体蛋白含量，研究了3个菌株的培养特性和碳源、氮源、pH值及温度对它们生长的影响。结果表明，MPC11、MPC12和MPA11三个菌株适于在BAP（有氮）固体培养基和BM+NZ和FMS液体培养基中生长。它们分别以葡萄糖、丙酮酸钠（含量均为1g／L）和吐温-80（含量为1ml／L）为唯一碳源或分别以酪蛋白水解物、酵母抽提物（含量均为0.5g／L）为唯一氮源时生长良好；能利用丙酸钠，但利用率低；且均不能利用尿素。MPCll和MPAll分离株适于在pHS.5一6.5范围内生长，pH7.0以上，菌体生长受到抑制。MPCll菌株在25℃一33℃生长良好，高于或低于此温度范围，菌株生长受到影响。不同菌株菌落颜色和形态、菌丝体颜色、生长速度因培养基种类的不同而有差异，且菌落和菌丝体的颜色也会随着培养时间的长短发生变化。 3.杨梅根瘤弗兰克氏菌株对杭生素和重金属的杭性及其机理。采用生长抑制分析法研究从杨梅根瘤分离的10个凡日。灯口菌株对9种重金属元素的杭性。一般来说，所有供试菌株对HgZ‘和Ag+敏感，大部分菌株对Pb，‘（5一1 ommol/L）、Cr207，一（5一10mmol/L）、As02一（5一10mmol/L）不敏感，部分菌株对Cu，‘、Co，’、Ni’‘和CdZ+也不敏感（Ml Cs>0.smm。l/L，有的菌株达到Sllunol/L）。菌株之间对各种重金属的杭性存在较大差异，其中菌株MDA21对所有供试重金属都敏感，而菌株MPAn几乎能抗所有供试重金属，且有几个菌株能同时杭多种重金属。低浓度的铅能促进抗铅菌株的生长，而高浓度的铅对其生长有一定的抑制;抗铅菌株在含铅的培养基中仍具有固氮酶活性，在一定浓度范围内，随着铅离子浓度的升高，固氮酶活性有所增强。Fr8nk，’a菌的杭铅机理可能是其具有结合或鳌合铅离子作用，对其他重金属的抗性机制尚不清楚。通过11个菌株对氨节青霉素、不.J福平和卡那霉素3种杭生素的敏感性试验，发现从杨梅根瘤分离的弗兰克氏菌株大多数能同时抗氨千青霉素和利福平，但对卡那霉素敏感。因此氨爷青霉素和利福平可作为筛选某些菌株的选择性标记。 4.高效固氮快生型Fr胡才1’a菌株的筛选。从固氮酶活性、菌株生长速度、侵染力和对杨梅幼苗生长的影响几方面综合考察了近40个杨梅根瘤Fr翻kz’口菌株，从中筛选出高效固氮快生型菌株5个和1个固氮活性高、侵染力较强、对杨梅生长促进作用明显但生长较慢的优良菌株，这6个菌株对杨梅幼苗的生长有明显的促进作用，新增株高、茎粗、鲜重分别比对照高17.23%²6.98%、25.”%-73.45%和150%一319.44%，其效果好于用杨梅根瘤直接接种。 5.杨梅弗兰克氏菌分离株的遗传多样性。对分离自杨梅根瘤的10株凡:刀kz’:菌分离株进行表型特性研究，并用特异引物扩增其165 rDNA，对扩增产物用2种限制性内切酶H1’刀厂I和施目n进行酶切，分析酶切图谱。结果表明:这10株斤:肪j己菌分离株不仅表型存在差异，而且其酶切图谱差异较大，说明基因型也有差异.即使从同一株革荞种杨梅根瘤中，不同时间分离获得的几翻打:菌分离株，表型和基因型也存在较大的差异，可能属于不同的基因种群。无论是从菌株表型分更多还原

【Abstract】 Myrica rubra Sieb. et Zucc (Chinese tree berry, red bayberry) is an important native fruit representing endemic species of China , is a tropical and subtropical fruit and is also a pioneer plant in Southern China. The root of M. rubra can fix nitrogen from atmosphere for plant growth in symbiosis with Frankia. It has extensive adaptability of cultivation andean easily be grown on barren lands with harsh environments and its trees have remarkable ecological effects which can increase water and soil conservation capability, adjust the climate of micro-environment, effectively control water flow and soil erosion, and decrease flood disaster. The trees of M. rubra also have a better fire resistibility to prevent fire from expanding. M. rubra is an ideal selection among the trees for soil conservation and forest ranges and has been selected as one kind of ecological tree by China National Forestry Bureau. With the adjustment of production structure and integrative development at mountainous regions, the area ofM.rubra has been increasing rapidly and M.rubra has been an important source of income for mountainous people.Nitrogen fixation is probably the second most important biochemical pathway after CO2 fixation on the earth. Biological nitrogen fixation is an important approach to solve lots of problems about population, food, energy and environment. It is more important for sustainable agriculture development to make full use of nitrogen fixation characteristics of M.rubra and how to exploit its economic and ecological benefits. Symbiotic nitrogen fixation,however, has less been reported. In order to exploit and develop efficiently symbiotic nitrogen fixation characteristics of M.rubra, the root nodules and its Frankia were studied thoroughly in the dissertation and the main results were summarized as followed./. Isolation and Identification of symbiotic nitrogen-fixing actinomycete from Myrica rubra root nodules Frankia strains were successfully isolated from M. rubra root nodules using Q++ and J-A solid media. Colonies of isolates on Q + + solid medium showed different colors and Gram-positive. Mycelia of the isolates are branched .septate and filamentous hypha about 0.6-1.1 μ m in diameter. The tip of hypha swells to form vesicle and these vesicles are eiher septate or non-septate, spherical and about 1.7~2.2 μ m in diameter. Sporangia of these isolates are pear-like or oval .about 15~30 μ m in diameter and spores are round , 1.4~1.7 μ m in diameter. The Frankia isolates grown in N-deficient BAP medium had acetylene reducing activities.2. Cultural characteristics of Frankia The cultural characteristics, and effects of carbon and nitrogen sources, pH, temperature on three Frankia strains were studied by microscopic examination and protein content detected by modified Bradford method. The results showed that BAP(nitrogen) solid media , BM+NZ and FMS liquid media are well suitable for the growth of Frankia MFC 11,MFC 12 and MPA11. They grow very well on BAP media with glucose .sodium pyruvate and Tween-80 as sole carbon source respectively and casamino acid or yeast extract as sole nitrogen source. They could use sodium propionate, but insufficiently. Urea could not be utilized. pH 5.5~ 6.5 were suitable for the growth of Frankia strain Mpcll and Mpall which were inhibited at pH above 7.0. Frankia MFC 11 grows normal under a temperature range of 25℃-33℃. The colors and morphologies of colonies, colors of mycelia and growth rates of different Frankia strains may be changed with the media types and cultural days.3. Heavy metal and antibiotic resistance of Frankia strains from root nodule of Myrica rubr The resistance of 10 Frankia strains to 9 kinds of heavy metals was determined by a growth inhibitionassay. In general, all of the strains were sensitive to low concentrations(<0.5 mmol/L= of Hg2+ and Ag+,but most of the strains were less sensitive to Pb2+ (5-10 mmol/L Cr2O72-(5-10 mmol/L) and AsO2- (5-10 mmol/L) while some of the strains were also less sensitive to Cu2+ Co2+ Ni2+更多还原