技术领域technical field
本发明涉及rsmA基因在植物根表定殖和促生方面的应用。The present invention relates to the application of rsmA gene in plant root surface colonization and growth promotion.
技术背景technical background
rsmA基因是在革兰氏阴性菌中广泛存在的转录后调节因子。在有些细菌的报道中，提到该基因参与微生物的中心碳代谢、菌体运动、生物膜及致病性等相关的生理过程，但是尚未见报道其在参与植物生长方面的功能。The rsmA gene is a post-transcriptional regulator ubiquitous in Gram-negative bacteria. In some bacterial reports, it is mentioned that this gene is involved in the physiological processes related to the central carbon metabolism of microorganisms, bacterial movement, biofilm and pathogenicity, but its function in participating in plant growth has not been reported yet.
微生物菌肥中使用的微生物多是从植物根际分离到的植物根际促生菌（PGPR），当这些促生菌在植物“根部定殖”，即在植物根表和近根土壤中稳定繁殖时，菌体可以利用植物根分泌的有机物作为碳源，例如有机酸，糖类，氨基酸等，这一独特的生态环境使得植物促生菌大量繁殖，形成非常高的菌密度，然后利用自身的生物固氮、溶磷解钾、分泌植物激素等方法促进植物生长的作用，实现植物-微生物互惠互利的高效共生关系。Most of the microorganisms used in microbial fertilizers are plant growth-promoting bacteria (PGPR) isolated from the rhizosphere of plants. When these growth-promoting bacteria colonize the "roots" of plants, they are stable in the root surface and near-root soil When multiplying, the bacteria can use the organic matter secreted by the plant roots as a carbon source, such as organic acids, sugars, amino acids, etc. This unique ecological environment makes the plant growth-promoting bacteria multiply, forming a very high bacterial density, and then using their own The methods of biological nitrogen fixation, phosphorus and potassium dissolution, and secretion of plant hormones can promote plant growth, and realize the efficient symbiotic relationship between plants and microorganisms.
固氮施氏假单胞菌A1501是我国科学家分离到的一株联合固氮菌。但是在田间环境下存在的众多的土著微生物会与A1501竞争氮素、碳素等营养物质。因此，大幅提高固氮施氏假单胞菌A1501在植物根部的定殖能力，使其在众多的根际微生物中获得竞争优势，大量定殖到植物根表并发挥促生作用，非常重要。Nitrogen-fixing Pseudomonas stutzeri A1501 is a combined nitrogen-fixing bacteria isolated by Chinese scientists. However, many indigenous microorganisms in the field environment will compete with A1501 for nutrients such as nitrogen and carbon. Therefore, it is very important to greatly improve the colonization ability of nitrogen-fixing Pseudomonas stutzeri A1501 in plant roots, so that it can gain a competitive advantage among numerous rhizosphere microorganisms, colonize a large number of plant root surfaces and play a role in promoting growth.
发明内容Contents of the invention
本发明的目的是寻找有关参与固氮施氏假单胞菌水稻根部定殖和促生相关的基因。The purpose of the present invention is to search for genes involved in the colonization and growth promotion of nitrogen-fixing Pseudomonas stutzeri rice roots.
本发明人在其实验室完成的A1501基因组中找到rsmA基因，该基因在A1501基因组中编号（Locustag）为PST1371，对应的蛋白编码在NCBI数据库编号是ABP79066.1。本发明首次发现了rsmA基因参与固氮施氏假单胞菌在植物根部定殖和促生方面的功能。研究表明，该基因的存在对微生物在根表的定向运动及高效定殖具有负调控作用，通过基因敲除、基因突变等有效手段使该基因的功能丧失后可以提高菌株在固体介质上的运动能力，并在植物根表产生更强的定殖能力，进而促进植物的生长。The inventor found the rsmA gene in the A1501 genome completed by his laboratory, the gene number (Locustag) in the A1501 genome is PST1371, and the corresponding protein code is ABP79066.1 in the NCBI database. The present invention discovers for the first time that the rsmA gene is involved in the colonization and growth promotion of nitrogen-fixing Pseudomonas stutzeri in plant roots. Studies have shown that the existence of this gene has a negative regulatory effect on the directional movement and efficient colonization of microorganisms on the root surface. The loss of the function of this gene through effective means such as gene knockout and gene mutation can improve the movement of the strain on solid media. ability, and produce stronger colonization ability on the plant root surface, thereby promoting plant growth.
即，rsmA基因作为负调控因子行使功能，当通过基因工程的手段使该基因的功能缺失后，获得的基因工程菌株具有更高的运动能力、生物膜合成能力以及更强的水稻根部定殖和促生能力。本发明涉及所述的突变菌的产生方法及其产生的菌株在微生物肥料产业中具有应用前景。That is, the rsmA gene functions as a negative regulatory factor. When the function of this gene is deleted by means of genetic engineering, the genetically engineered strains obtained have higher motility, biofilm synthesis ability, and stronger rice root colonization and Growth-promoting ability. The invention relates to the production method of the mutant bacteria and the produced strains have application prospects in the microbial fertilizer industry.
基因组分析表明，在固氮施氏假单胞菌A1501中存在一个跟大肠杆菌csrA的氨基酸序列87%的相似度，和铜绿假单胞菌rsmA基因氨基酸序列同源性高达97%的类似物。我们通过同源重组的方法对固氮假单胞菌中的rsmA基因进行整合突变使其功能缺失。结果发现，突变株在固体培养基上菌落形态发生明显的变化，菌落变得干燥，不易从培养基上剥离。野生型A1501在含0.5%琼脂的LB平板上，几乎不发生swarm运动，但RsmA突变株的swarm运动能力极大的增强，此外，RsmA的突变导致了菌体生物薄膜生成量明显增加。此外，通过接种野生型及突变株菌株于水稻幼苗根部，结果发现，rsmA功能的缺失导致了固氮假单胞菌在水稻根部定殖能力和促生能力都大幅增强。Genome analysis showed that in nitrogen-fixing Pseudomonas stutzeri A1501, there was an amino acid sequence similarity of 87% to Escherichia coli csrA, and an analogue with an amino acid sequence homology of up to 97% of the rsmA gene of Pseudomonas aeruginosa. We used the method of homologous recombination to integrate the rsmA gene in Pseudomonas nitrogen-fixing bacteria to make it lose its function. It was found that the colony morphology of the mutant strains changed significantly on the solid medium, and the colonies became dry and difficult to peel off from the medium. The wild-type A1501 swarm movement hardly occurred on the LB plate containing 0.5% agar, but the swarm movement ability of the RsmA mutant strain was greatly enhanced. In addition, the mutation of RsmA led to a significant increase in the production of bacterial biofilm. In addition, by inoculating wild-type and mutant strains in the roots of rice seedlings, it was found that the loss of rsmA function led to a significant increase in the colonization and growth-promoting abilities of Pseudomonas nitrogen-fixing bacteria in rice roots.
本发明人通过对PseudomonasstutzeriA1501的全基因组序列和基因注释分析，发现构建rsmA基因功能缺失的突变株可以用于生产增强固氮作用的微生物菌肥。Through the analysis of the whole genome sequence and gene annotation of PseudomonasstutzeriA1501, the present inventors found that the construction of a mutant strain lacking the function of the rsmA gene can be used to produce microbial fertilizers that enhance nitrogen fixation.
上述构建rsmA基因功能缺失的突变株可以采用多种形式，如整合突变方式，也可以采用缺失突变，点突变等方式，只要能够使rsmA基因功能缺失就可以得到相似的表型和实验结果。The above-mentioned construction of mutants lacking the function of the rsmA gene can adopt various forms, such as integration mutation, deletion mutation, point mutation, etc., as long as the function of the rsmA gene can be lost, similar phenotypes and experimental results can be obtained.
在本发明的一个实例中，采用同源重组的方式对rsmA基因进行整合突变，获得了rsmA基因功能缺失突变株。本发明对所得到的上述rsmA突变株进行了如下测定，证明其可高效定殖水稻，小麦，玉米等禾本科农作物根部，并对植物有促生长作用：In an example of the present invention, the rsmA gene is integrated and mutated by means of homologous recombination to obtain a mutant strain of rsmA gene function loss. The present invention has carried out the following measurements on the obtained above-mentioned rsmA mutant strain, which proves that it can efficiently colonize the roots of gramineous crops such as rice, wheat, and corn, and has a growth-promoting effect on plants:
1）菌体swarming运动1) Cell swarming movement
即，测定菌体在琼脂含量为0.4%-0.7%的半固体培养基表面上运动扩散，发现突变株在固体界面上的swarming运动能力显著增强，这可以导致菌体在水稻根表定殖后，通过swarming运动形式从定殖初始位点向周围扩张扩大定殖的面积，使菌体广泛占据植物的根表，获得更好的营养来源，在与其他土著微生物的竞争中占据先机。That is, the movement and diffusion of the bacteria on the surface of the semi-solid medium with agar content of 0.4%-0.7% was measured, and it was found that the swarming ability of the mutant strain on the solid interface was significantly enhanced, which could lead to the growth of the bacteria after colonization on the root surface of rice. , expand the area of colonization from the initial site of colonization to the surrounding area through the form of swarming movement, so that the bacteria can widely occupy the root surface of plants, obtain better nutrient sources, and take the lead in the competition with other indigenous microorganisms.
2）生物膜形成（细菌在固体表面形成一种具有一定三维空间结构的多细胞结构，被称为生物膜），发现突变株生物膜的生成能力增强，证明rsmA的突变促进了施氏假单胞菌生物膜的形成。生物膜的形成对于固氮微生物是非常重要的，形成了生物膜这种特殊的结构，微生物的生长、固氮等多种生理反应才能够被激活并稳定进行。2) Biofilm formation (bacteria form a multicellular structure with a certain three-dimensional spatial structure on a solid surface, called a biofilm), and it was found that the biofilm generation ability of the mutant strain was enhanced, which proved that the mutation of rsmA promoted the Schneider pseudomonas Bacterial biofilm formation. The formation of biofilm is very important for nitrogen-fixing microorganisms. Only when the special structure of biofilm is formed, various physiological reactions such as microbial growth and nitrogen fixation can be activated and carried out stably.
3）水稻根表竞争性定殖实验3) Rice root surface competitive colonization experiment
在突变株在和固氮假单胞菌A1501野生型的水稻根际竞争性定殖实验中，发现rsmA突变株的定殖能力高出野生型10倍以上；In the rice rhizosphere competitive colonization experiment between the mutant strain and Pseudomonas nitrogen-fixing A1501 wild type, it was found that the colonization ability of the rsmA mutant strain was more than 10 times higher than that of the wild type;
4）水稻促生实验4) Rice growth promotion experiment
在实验室环境下，采用野生型A1501及rsmA突变株接种水稻植株，在对水稻的促生效果上，无论水稻株高还是植株地上部分与地下部分的干重上都具有明显的优势。In the laboratory environment, using wild-type A1501 and rsmA mutant strains to inoculate rice plants has obvious advantages in promoting the growth of rice, regardless of the height of the rice plant or the dry weight of the above-ground and underground parts of the plant.
5）固氮酶活测定5) Determination of Nitrogenase Activity
实验发现：rsmA突变株的固氮酶活相对于野生型虽然没有发生明显改变，说明rsmA的缺失对于固氮效率没有产生影响。The experiment found that the nitrogenase activity of the rsmA mutant strain did not change significantly compared with the wild type, indicating that the absence of rsmA had no effect on nitrogen fixation efficiency.
附图说明Description of drawings
图1是rsmA整合突变PCR验证，图中：M为trans2Kplus2DNAmarker，1为利用pK18mobcon-F与rsmA下游-R为引物，rsmA插入突变株基因组为模板扩增出的800bp片段；Figure 1 is the PCR verification of the rsmA integration mutation. In the figure: M is trans2Kplus2DNAmarker, 1 is the 800bp fragment amplified using pK18mobcon-F and rsmA downstream-R as primers, and the rsmA insertion mutant genome as a template;
图2是A1501野生型和rsmA突变株固氮酶活的测定；Fig. 2 is the determination of nitrogenase activity of A1501 wild type and rsmA mutant strain;
图3是A1501和rsmA突变株在固态培养基上的swarming能力测定，其中，图左是rsmA基因突变株图右是野生型A1501；Figure 3 is the determination of the swarming ability of A1501 and rsmA mutant strains on solid medium, where the left side of the figure is the rsmA gene mutant strain and the right side is wild-type A1501;
图4是A1501和rsmA突变株生物膜生成能力测定对比，rsmA突变株形成的菌膜明显要比野生型的要厚。Figure 4 is a comparison of the biofilm formation ability of A1501 and rsmA mutant strains. The biofilm formed by the rsmA mutant strain is obviously thicker than that of the wild type.
具体实施方式detailed description
下面结合具体实施例，进一步阐述本发明。这些实施例仅用于举例说明本发明的方法，而不用于限制本发明的范围。凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件。Below in conjunction with specific embodiment, further illustrate the present invention. These examples are only used to illustrate the method of the present invention and are not intended to limit the scope of the present invention. Where specific experimental conditions are not indicated, conventional conditions well known to those skilled in the art are followed.
实施例1以施氏假单胞菌A1501为出发菌株构建rsmA突变株Embodiment 1 takes Pseudomonas stutzeri A1501 as the starting strain to construct rsmA mutant strain
本实验构建的rsmA突变株采用的是整合突变的方法。基本的原理为DNA同源重组。The rsmA mutant strain constructed in this experiment adopts the method of integration mutation. The basic principle is DNA homologous recombination.
实验方法：experimental method:
（1）：采用PCR扩增法获得截短的rsmA基因部分片段，命名为rsmA’，将rsmA’片段连接到pMD-19T克隆载体上，对rsmA’进行DNA测序，确保序列的真实性。(1): The truncated rsmA gene fragment was obtained by PCR amplification method, named rsmA', and the rsmA' fragment was connected to the pMD-19T cloning vector, and DNA sequencing was performed on rsmA' to ensure the authenticity of the sequence.
（2）：对rsmA’克隆载体进行限制性双酶切，将切下的rmsA’连接到pK18mob质粒上，获得rsmA’自杀质粒。(2): Digest the rsmA' cloning vector with restriction enzymes, and connect the excised rmsA' to the pK18mob plasmid to obtain the rsmA' suicide plasmid.
（3）：通过三亲结合方法，rsmA’自杀质粒导入到A1501中，利用A15限制性培养基和卡那霉素进行筛选。(3): The rsmA' suicide plasmid was introduced into A1501 by the method of triparental binding, and the A15 restriction medium and kanamycin were used for screening.
（4）：质粒上的rsmA’和A1501基因组上的rsmA发生同源单交换。利用pK18mob上的通用引物pK18mobconF和rsmA下游DNA片段的反向引物进行菌落PCR扩增。能扩增出大小约800bp的条带的菌株已经发生了同源交换。(4): rsmA' on the plasmid and rsmA on the A1501 genome undergo homologous single exchange. Colony PCR amplification was performed using the universal primer pK18mobconF on pK18mob and the reverse primer for the DNA fragment downstream of rsmA. Homologous exchange has occurred in the strain that can amplify a band with a size of about 800bp.
实验结果：Experimental results:
利用pK18mob上的通用引物pK18mobconF和rsmA下游DNA片段的反向引物进行菌落PCR扩增，能扩增出大小约800bp的条带（如图1）。说明已经发生了同源交换，获得rsmA突变株。Using the general primer pK18mobconF on pK18mob and the reverse primer of the downstream DNA fragment of rsmA to perform colony PCR amplification, a band with a size of about 800 bp can be amplified (as shown in Figure 1). It shows that homologous exchange has occurred, and the rsmA mutant strain has been obtained.
实施例2野生型A1501和rsmA突变株的swarming运动Example 2 The swarming movement of wild-type A1501 and rsmA mutant strain
实验方法：experimental method:
（1）从LB平板上挑取新鲜活化的单菌落，接种到含20mLLB液体培养基的50mL三角瓶中，rsmA突变株培养基需要加入50μg/mL的卡那霉素，30℃，200rpm培养过夜。(1) Pick a freshly activated single colony from the LB plate and inoculate it into a 50mL Erlenmeyer flask containing 20mL of LB liquid medium. The rsmA mutant medium needs to be added with 50μg/mL of kanamycin, and cultivate overnight at 30°C and 200rpm .
（2）吸取1%过夜菌培养物接种到含20mLLB液体培养基的50mL三角瓶中。rsmA突变株培养基需要加入50μg/mL的卡那霉素。30℃，200rpm培养12小时，测定菌体密度OD600≈2.5。(2) Take 1% of the overnight bacterial culture and inoculate it into a 50mL Erlenmeyer flask containing 20mL of LB liquid medium. The rsmA mutant culture medium needs to add 50 μg/mL kanamycin. Cultivate at 30°C and 200 rpm for 12 hours, and measure the cell density OD600≈2.5.
（3）在超净工作台中吸取2μL菌液滴在已经放置过夜的含0.5%琼脂的LB固体培养基平板的中心点上。待菌液自然干燥后，倒置平板，30℃静置培养24-48小时。对实验结果进行照相记录。(3) Pipette 2 μL of the bacteria drop onto the center point of the LB solid medium plate containing 0.5% agar that has been placed overnight in the ultra-clean workbench. After the bacterial liquid dries naturally, invert the plate and incubate at 30°C for 24-48 hours. Photographic records were made of the experimental results.
实验结果：Experimental results:
见图3。Swarming运动是菌体在固体表面靠鞭毛向周围扩张的运动形式。在固氮施氏假单胞菌野生型中swarming运动能力很弱，而rsmA突变株的swarming能力大大增强。并且rsmA突变株形成的菌落表面有褶皱，菌落边缘不规则，菌落的湿润程度要比野生型低，形成的菌膜更加致密。这可能是细胞表面性质发生变化造成的。Swarming运动能力增强可以使得菌体在水稻根表定殖后，从定殖位点向周围扩张，从而增加菌体在水稻根表的定殖面积。See Figure 3. Swarming movement is a form of movement in which bacteria expand to the surrounding area by flagella on a solid surface. The swarming ability of the nitrogen-fixing Pseudomonas stutzeri wild type was weak, but the swarming ability of the rsmA mutant was greatly enhanced. And the surface of the colony formed by the rsmA mutant strain was wrinkled, the edge of the colony was irregular, the degree of humidity of the colony was lower than that of the wild type, and the formed biofilm was denser. This may be due to changes in the properties of the cell surface. The enhancement of Swarming ability can make the bacteria expand from the colonization site to the surrounding area after colonization on the rice root surface, thereby increasing the colonization area of the bacteria on the rice root surface.
实施例3野生型A1501和rsmA突变株的生物膜形成Example 3 Biofilm formation of wild-type A1501 and rsmA mutant strains
实验方法：experimental method:
（1）从LB平板上挑取新鲜活化的单菌落，接种到含20mLLB液体培养基的50mL三角瓶中，rsmA突变株培养基需要加入50μg/mL的卡那霉素，30℃，200rpm培养过夜。(1) Pick a freshly activated single colony from the LB plate and inoculate it into a 50mL Erlenmeyer flask containing 20mL of LB liquid medium. The rsmA mutant medium needs to be added with 50μg/mL of kanamycin, and cultivate overnight at 30°C and 200rpm .
（2）吸取1%过夜菌培养物接种到含20mLLB液体培养基的50mL三角瓶中。rsmA突变株培养基需要加入50μg/mL的卡那霉素。30℃，200rpm培养12小时，测定菌体密度OD600≈2.5。(2) Take 1% of the overnight bacterial culture and inoculate it into a 50mL Erlenmeyer flask containing 20mL of LB liquid medium. The rsmA mutant culture medium needs to add 50 μg/mL kanamycin. Cultivate at 30°C and 200 rpm for 12 hours, and measure the cell density OD600≈2.5.
（3）将10mL菌培养物4℃4000g离心10分钟，弃掉上清液，利用生理盐水悬浮菌体，再次4000g离心10分钟离心。弃掉上清液，利用A15限制性培养液悬浮菌体，调节OD600=1.0。(3) Centrifuge 10 mL of bacterial culture at 4000g for 10 minutes at 4°C, discard the supernatant, suspend the bacteria with physiological saline, and centrifuge again at 4000g for 10 minutes. Discard the supernatant, use the A15 restricted medium to suspend the cells, and adjust the OD600 =1.0.
（4）吸取5mL上述菌悬液加入到20×150mm的带盖玻璃试管中。30℃静置培养48小时。观察试管内液体和空气交界处菌膜的形成情况。(4) Take 5mL of the above bacterial suspension and add it to a 20×150mm glass test tube with a cover. Incubate at 30°C for 48 hours. Observe the formation of bacterial film at the junction of liquid and air in the test tube.
（5）小心倒掉菌液，轻轻加入蒸馏水洗涤试管，倒掉蒸馏水，重复一次。加入5mL1%的结晶紫染液静置5分钟，轻柔倒掉染液，利用蒸馏水轻柔洗涤试管两次，拍照记录。(5) Carefully pour off the bacterial solution, gently add distilled water to wash the test tube, pour off the distilled water, and repeat once. Add 5mL of 1% crystal violet dyeing solution and let it stand for 5 minutes, pour off the dyeing solution gently, wash the test tube twice with distilled water gently, take pictures and record.
实验结果：Experimental results:
见图4。在A1501野生型和rsmA突变株的玻璃试管液体和空气交界处都能形成一层白色褶皱菌膜，rsmA突变株形成的菌膜明显要比野生型的要厚。同时在两者的试管管壁菌液最上沿会形成一个菌圈，但rsmA突变株的菌圈明显要厚的多。See Figure 4. A layer of white wrinkled biofilm could be formed at the interface between the liquid and the air in the glass test tube of A1501 wild type and rsmA mutant strain, and the biofilm formed by the rsmA mutant strain was obviously thicker than that of the wild type. At the same time, a bacterial circle will be formed on the uppermost edge of the test tube wall bacterial solution, but the bacterial circle of the rsmA mutant strain is obviously much thicker.
实验结论：Experimental results:
rsmA的突变促进了施氏假单胞菌生物膜的形成。Mutations in rsmA promote Pseudomonas stutzeri biofilm formation.
实施例4野生型A1501和rsmA突变株的水稻根部竞争性定殖试验Example 4 Competitive colonization test of rice roots of wild-type A1501 and rsmA mutant strains
实验方法：experimental method:
（1）水稻种子表面消毒。将水稻种子利用蒸馏水漂洗两遍，去除种子表面的灰尘，并且去除干瘪的水稻种子。70%酒精洗涤种子2分钟，10%的次氯酸钠洗涤15分钟，在洗涤过程中，放到摇床上缓慢摇动。最后利用无菌蒸馏水漂洗5-6遍。(1) Surface disinfection of rice seeds. Rinse the rice seeds twice with distilled water to remove the dust on the surface of the seeds and remove the shriveled rice seeds. Wash the seeds with 70% alcohol for 2 minutes, and wash with 10% sodium hypochlorite for 15 minutes. During the washing process, put them on a shaker and shake slowly. Finally, rinse with sterile distilled water 5-6 times.
（2）将消毒后的水稻种子放入含有无菌水和纱布的培养皿。30℃催芽4天。(2) Put the sterilized rice seeds into a Petri dish containing sterile water and gauze. Germinate at 30°C for 4 days.
（3）挑选发芽情况一致5棵水稻幼苗转移到含有20mL水稻培养液的大试管中，每个实验组设三个平行组。将水稻幼苗转移到人工气候培养箱中。27℃，12小时光照，12小时黑暗，相对湿度70%。培养48小时。(3) Select 5 rice seedlings with the same germination status and transfer them to a large test tube containing 20 mL of rice culture medium, and set up three parallel groups for each experimental group. Transfer the rice seedlings to an artificial climate incubator. 27°C, 12 hours light, 12 hours dark, relative humidity 70%. Incubate for 48 hours.
（4）从LB平板上挑取新鲜活化的单菌落，接种到含20mLLB液体培养基的50mL三角瓶中，rsmA突变株培养基需要加入50μg/mL的卡那霉素，30℃，200rpm培养过夜。(4) Pick a freshly activated single colony from the LB plate and inoculate it into a 50mL Erlenmeyer flask containing 20mL of LB liquid medium. The rsmA mutant medium needs to be added with 50μg/mL of kanamycin, and cultivate overnight at 30°C and 200rpm .
（5）吸取1%过夜菌培养物接种到含20mLLB液体培养基的50mL三角瓶中。rsmA突变株培养基需要加入50μg/mL的卡那霉素。30℃，200rpm培养12小时，测定菌体密度OD600≈2.5。(5) Take 1% of the overnight bacterial culture and inoculate it into a 50mL Erlenmeyer flask containing 20mL of LB liquid medium. The rsmA mutant culture medium needs to add 50 μg/mL kanamycin. Cultivate at 30°C and 200 rpm for 12 hours, and measure the cell density OD600≈2.5.
（6）将10mL菌培养物4℃4000g离心10分钟，弃掉上清液，利用生理盐水悬浮菌体，再次4000g离心10分钟离心。弃掉上清液，利用生理盐水悬浮菌体，调节OD600=1.0。(6) Centrifuge 10 mL of bacterial culture at 4000g for 10 minutes at 4°C, discard the supernatant, suspend the bacteria with physiological saline, and centrifuge again at 4000g for 10 minutes. Discard the supernatant, suspend the cells with physiological saline, and adjust OD600 =1.0.
（7）向含有水稻幼苗的大试管中加入2.5mL的A1501菌悬液和2.5mL的rsmA菌悬液，混匀，此时菌体总密度为OD600≈0.2。27℃，12小时光照，12小时黑暗，相对湿度70%。培养48小时。(7) Add 2.5mL of A1501 bacterial suspension and 2.5mL of rsmA bacterial suspension to the large test tube containing rice seedlings, and mix well. At this time, the total density of bacteria is OD600≈0.2. Hours of darkness, 70% relative humidity. Incubate for 48 hours.
（8）从上述培养试管中取出长势相近的3棵水稻。在超净工作台中用无菌蒸馏水轻轻仔细冲洗根部，利用滤纸将根部水分吸干，重复冲洗根部2次。将根部剪下，放入含有1mL蒸馏水的1.5mL离心管中，利用涡旋仪剧烈震荡5分钟，将所得菌液按10倍梯度稀释。各梯度吸取100微升液体涂布在含有四环素或卡那霉素的A15限制性固体平板上。30℃静置培养48个小时，对平板上的单菌落进行计数，计数时选取菌落数为30-300个平板进行计数。将震荡完成的水稻根取出，用滤纸吸干水分。80℃烘箱烘烤1小时。利用分析天平称取水稻根干重。(8) Take out 3 rice plants with similar growth from the above culture test tubes. Gently and carefully rinse the roots with sterile distilled water in an ultra-clean workbench, use filter paper to dry the roots, and repeat the rinse twice. Cut off the root, put it into a 1.5mL centrifuge tube containing 1mL of distilled water, shake vigorously for 5 minutes with a vortexer, and dilute the obtained bacterial solution in a 10-fold gradient. 100 microliters of each gradient was pipetted onto A15-restricted solid plates containing tetracycline or kanamycin. After static culture at 30°C for 48 hours, count the single colonies on the plate, and select plates with 30-300 colonies for counting. The rice roots that have been shaken are taken out, and the water is blotted dry with filter paper. Bake in an oven at 80°C for 1 hour. The dry weight of rice roots was weighed using an analytical balance.
菌体定殖密度计算公式为c=(菌落数×稀释倍数×10)÷水稻根干重(g)The formula for calculating the bacterial colonization density is c=(number of colonies×dilution factor×10)÷dry weight of rice roots (g)
实验结果：Experimental results:
A1501的定殖密度为2.34×107cfu/根干重（g），rsmA突变株的定殖密度为3.60×108cfu/根干重（g），为野生型A1501的15.4倍。The colonization density of A1501 was 2.34×107 cfu/root dry weight (g), and the colonization density of the rsmA mutant was 3.60×108 cfu/root dry weight (g), which was 15.4 times that of wild-type A1501.
实验结论：Experimental results:
rsmA突变株的竞争性定殖能力比野生型A1501有了很大的提高。The competitive colonization ability of the rsmA mutant was greatly improved compared with the wild-type A1501.
实施例5野生型A1501和rsmA突变株对水稻幼苗的促生试验Embodiment 5 Wild-type A1501 and rsmA mutant strain to the growth-promoting experiment of rice seedling
实验方法：experimental method:
（1）水稻种子消毒、催芽及转接参照本章实施例5。(1) Disinfection, germination and transfer of rice seeds refer to Example 5 in this chapter.
（2）挑选发芽情况一致的50株水稻幼苗转移到含有200mL水稻培养液的1L的大烧杯中，分别加入20mLOD600为1.0野生型A1501及rsmA突变株菌液。。27℃条件下，光照条件为12小时光照，12小时黑暗，相对湿度70%，培养10天，将幼苗取出，测量水稻的株高(cm)，将水稻根剪下，蒸馏水洗涤2次。滤纸吸干根表水分后将水稻根80℃烘箱烘烤1小时。称量水稻根干重。(2) Select 50 rice seedlings with the same germination status and transfer them to a 1L large beaker containing 200mL rice culture solution, and add 20mL OD600 of 1.0 wild-type A1501 and rsmA mutant strain bacterial solution respectively. . Under the condition of 27°C, the light conditions were 12 hours of light, 12 hours of darkness, and a relative humidity of 70%. After culturing for 10 days, the seedlings were taken out, the plant height (cm) of the rice was measured, the roots of the rice were cut off, and washed twice with distilled water. After absorbing the water on the root surface with filter paper, bake the rice root in an oven at 80° C. for 1 hour. Weigh the dry weight of rice roots.
实验结果：10天后，rsmA突变株无论在株高上还是地上干重与地下干重上都具有明显的优势（见表1）。接种rsmA突变株的水稻株高平均达到23.4±1.6cm，而野生型菌株高为16.8±1.4cm，而不接种任何菌剂的对照株高平均达到14.6±1.2cm。Experimental results: After 10 days, the rsmA mutant had obvious advantages in terms of plant height, aboveground dry weight and underground dry weight (see Table 1). The average plant height of the rice inoculated with the rsmA mutant reached 23.4±1.6 cm, while the height of the wild-type strain was 16.8±1.4 cm, and the average plant height of the control without any inoculation agent reached 14.6±1.2 cm.
表1野生型A1501及rsmA突变株对水稻的促生能力测定Table 1 Determination of the growth-promoting ability of wild-type A1501 and rsmA mutant strains on rice
实验结论：Experimental results:
rsmA突变株比野生型A1501具有更强的植物促生能力。The rsmA mutant strain had stronger plant growth-promoting ability than wild-type A1501.
实施例6野生型A1501和rsmA突变株的固氮酶活测定Nitrogenase activity assay of embodiment 6 wild-type A1501 and rsmA mutant strain
实验方法：experimental method:
（1）将待测的菌株从LB平板上接种到含5mLLB液体培养基的试管中，30℃，200rpm摇床培养过夜。(1) Inoculate the strain to be tested from the LB plate into a test tube containing 5 mL of LB liquid medium, and cultivate overnight at 30°C on a shaker at 200 rpm.
（2）吸取1mL过夜培养物加入到含20mLLB培养液的三角瓶中，30℃，200rpm培养3-4小时，此时菌体OD600大约1.0。(2) Take 1 mL of the overnight culture and add it to a Erlenmeyer flask containing 20 mL of LB culture solution, and incubate at 30°C and 200 rpm for 3-4 hours. At this time, the OD600 of the bacteria is about 1.0.
（3）利用50mL离心管5000rpm离心8分钟，倒掉上清液，加入大约30mL的A15无氮限制性培养基悬浮菌沉淀。5000rpm离心8分钟，倒掉上清液，再次加入30mL的A15无氮限制性培养基悬浮菌沉淀。倒掉上清液，加适量的A15无氮限制性培养基悬浮菌体，测定OD600，稀释到1.0。(3) Use a 50mL centrifuge tube to centrifuge at 5000rpm for 8 minutes, discard the supernatant, and add about 30mL of A15 nitrogen-free limited medium to suspend the bacterial pellet. Centrifuge at 5000rpm for 8 minutes, pour off the supernatant, and add 30mL of A15 nitrogen-free limited medium again to suspend the bacterial pellet. Pour off the supernatant, add an appropriate amount of A15 nitrogen-free limited medium to suspend the bacteria, measure the OD600 , and dilute to 1.0.
（4）在无菌的窄口三角瓶中加入9mL的A15无氮限制性培养基和1mL菌液。塞好橡皮塞。每个菌株设立5个重复。(4) Add 9mL of A15 nitrogen-free limiting medium and 1mL of bacterial solution into a sterile narrow-mouth Erlenmeyer flask. Insert the rubber stopper. Five replicates were set up for each strain.
（5）利用氩气对窄口三角瓶中的空气进行排空，排气时间5分钟。利用微量取样器向每个瓶中注入1%的氧气，和10%的乙炔。30℃，200rpm培养。(5) Use argon to evacuate the air in the narrow mouth Erlenmeyer flask for 5 minutes. Inject each vial with 1% oxygen and 10% acetylene using a microsampler. Cultivate at 30°C, 200rpm.
（6）培养四小时后取样。利用微量取样器吸取0.25mL的三角瓶气体。注入气相色谱仪中，记录乙烯和乙炔的峰面积。每小时取样进行测量。(6) Samples were taken after four hours of incubation. Use a micro sampler to draw 0.25mL of gas in the Erlenmeyer flask. Inject into a gas chromatograph and record the peak areas of ethylene and acetylene. Samples are taken hourly for measurement.
（7）利用考马斯亮蓝法测定三角瓶中菌液的全蛋白含量。(7) Determination of the total protein content of the bacterial liquid in the Erlenmeyer flask by the Coomassie brilliant blue method.
（8）利用公式固氮酶活=乙烯峰面积*（三角瓶的气相总体积/取样体积）/（1nmol乙烯标准峰面积*反应时间*菌体全蛋白总量）。其中1nmol乙烯标准峰面积=1926；菌体全蛋白总量为0.568mg。(8) Use the formula nitrogenase activity = ethylene peak area * (total gas phase volume of the triangular flask / sampling volume) / (1nmol ethylene standard peak area * reaction time * total bacterial total protein). Among them, the standard peak area of 1nmol ethylene = 1926; the total protein of the bacteria is 0.568mg.
实验结果：Experimental results:
固氮假单胞菌rsmA突变株的固氮酶活相比野生型没有发生明显的变化（如图2）。The nitrogenase activity of the Pseudomonas nitrogenase rsmA mutant strain did not change significantly compared with the wild type (Figure 2).
实验结论：Experimental results:
rsmA突变株比野生型A1501具有更强的植物促生能力并不是通过提高固氮酶活，而是通过其它途径或方式。The stronger plant growth-promoting ability of the rsmA mutant than the wild-type A1501 is not through increasing nitrogenase activity, but through other pathways or methods.
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