黄腐酸提高苜蓿产量及促进Sinorhizobium meliloti与苜蓿结瘤固氮的作用机理

发布时间：2018-04-09 03:27

  本文选题：紫花苜蓿　切入点：苜蓿中华根瘤菌　出处：《中国农业大学》2016年博士论文

【摘要】：苜蓿是一种具有很高经济价值和营养价值的多年生豆科牧草,目前苜蓿产量已经严重供不应求,限制了我国畜牧业发展。接种与苜蓿品种相匹配的高效根瘤菌不仅可以提高苜蓿产量和品质,而且可以减少氮肥使用,提高土壤肥力。本实验室前期研究发现微生物降解褐煤产生的黄腐酸对多种禾本科作物都有很好的增产效果,而且能够促进大豆结瘤固氮,显著提高大豆产量。本课题研究了两株苜蓿高效根瘤菌Sinorhizibium meliloti CCBAU01199和Sinorhizibium meliloti CCBAU01290与中苜一号的共生有效性、黄腐酸对苜蓿产量的影响、黄腐酸对苜蓿—根瘤菌体系的刺激作用及其机理,并针对内蒙地区的八个苜蓿品种进行了根瘤菌的分离及高效菌株的筛选,为提高苜蓿—根瘤菌体系的固氮效率,大力推广苜蓿种植中接种根瘤菌提供理论和材料基础。田间试验结果表明,接种两株根瘤菌均能够显著提高苜蓿产量,而且S. meliloti CCBAU01199的增产效果高于S. meliloti CCBAU01290,说明S. meliloti CCBAU01199与中苜一号的匹配性更好。根瘤菌不同的接种方式也显著影响接种效果,S. meliloti CCBAU01199最佳的接种方式是拌种,连续两年使苜蓿产量分别提高了37.57%和24.78%。对土壤理化性质测定结果表明,苜蓿接种根瘤菌提高了土壤全氮、碱解氮及有机质含量,但使土壤有效磷和速效钾含量降低,所以在苜蓿种植过程中需补施磷肥和钾肥。喷施0.5g/L黄腐酸对苜蓿的增产效果与接种高效根瘤菌相似,连续两年使苜蓿产量分别提高了29.57%和20.34%,接种S. meliloti CCBAU01290并喷施黄腐酸比只接种根瘤菌苜蓿增产9.16%。检测苜蓿结瘤情况发现黄腐酸显著提高了土著根瘤菌及接种根瘤菌与苜蓿的结瘤数、根瘤鲜重及固氮酶活。以苜蓿中华根瘤菌模式菌株S. meliloti 1021为对象研究了黄腐酸促进S. meliloti与苜蓿结瘤固氮的作用机理。结果发现,黄腐酸显著促进了S. meliloti 1021的生长,使其细胞密度提高了5.07倍,而且使具有完整群体感应系统的菌株S. meliloti 8530 (1021,expR+)的细胞密度提高了20～50倍,说明黄腐酸的调控作用和群体感应系统有关。实时荧光定量PCR(RT-qPCR)的结果显示黄腐酸显著抑制了S. meliloti 8530的群体感应相关基因expR、sinlI、sinR的表达,而且黄腐酸主要是通过抑制expR基因的表达而调控其群体感应系统。对黄腐酸的作用模式进行比较分析,发现黄腐酸可能是通过S. meliloti 8530中的QsrR蛋白起作用。对qsrR基因进行敲除证实了上述假设,在meliloti △qsrR突变株中,expR基因的表达上调,而且其表达不再受黄腐酸的抑制。凝胶阻滞及细菌单杂交实验证明QsrR蛋白能够与expR基因启动子结合而直接抑制expR基因的表达。而且黄腐酸通过加强QsrR蛋白与expR基因启动子的结合而进一步抑制expR基因的表达。本研究首次鉴定了一个在转录水平调控expR基因表达的蛋白—QsrR,证明了黄腐酸通过QsrR抑制S. meliloti8530的群体感应系统而促进其生长,抑制其EPSⅡ及AHLs的合成,有利于根瘤菌在根圈的定殖并减弱宿主的防御反应,从而有利于共生固氮。研究结果为提高共生固氮效率奠定了重要基础。从内蒙科尔沁地区采集根瘤共分离得到S. meliloti 74株。针对科尔沁地区的八个苜蓿品种进行高效根瘤菌的匹配实验,初步为每个苜蓿品种筛选到1～3株高效根瘤菌。为科尔沁沙地地区的苜蓿接种根瘤菌积累了优良菌株,也丰富了苜蓿高效根瘤菌菌种资源。
[Abstract]:Alfalfa is a kind of high economic value and nutritional value of perennial legume, the yield of alfalfa has a serious shortage, limiting the development of animal husbandry in China. High efficient Rhizobium inoculation and alfalfa varieties matching can not only improve the yield and quality of alfalfa, but also can reduce nitrogen use, improve soil fertility. In our previous study discovery of fulvic acid from lignite degraded by microbial have good effect on increasing variety of gramineous crops, but also can promote soybean nodulation, significantly increased the yield of soybean. This study effectiveness of two strains of Alfalfa Rhizobium Sinorhizibium meliloti CCBAU01199 and Sinorhizibium meliloti CCBAU01290 and Zhongmu No.1, Huang humic acid on alfalfa yield, stimulation effect and mechanism of fulvic acid on Alfalfa Rhizobia system, and for the eight Inner Mongolia area Alfalfa varieties were screened for strains isolated and efficient strains, in order to improve the efficiency of nitrogen fixation of Alfalfa Rhizobia system, and vigorously promote the Rhizobium inoculation of alfalfa planting to provide theoretical and material basis. Field test results show that two strains of Rhizobium inoculation could significantly improve yield of alfalfa, and the S. meliloti CCBAU01199 yield S. meliloti CCBAU01290 is higher than that of S., CCBAU01199 and meliloti, better Zhongmu No.1. Inoculation of Rhizobium inoculation also significantly influence the different effect of S. meliloti CCBAU01199 the best inoculation method with two consecutive years, the yield of alfalfa were increased by 37.57% and 24.78%. on soil physicochemical properties determination results showed that inoculation of Alfalfa bacteria increased soil total nitrogen, available nitrogen and organic matter content, but the soil available phosphorus and potassium content decreased, so in alfalfa planting The need to fill the application of P and K fertilizer. In the process of spraying 0.5g/L fulvic acid on Alfalfa Yield and inoculating rhizobia is similar to that for two consecutive years of alfalfa yield were increased by 29.57% and 20.34%, meliloti and CCBAU01290 were inoculated with S. fulvic acid than Rhizobium inoculation of alfalfa was increased by 9.16. Detection of nodulation found yellow rot acid significantly increased the number of nodules of indigenous rhizobia and inoculation of rhizobia and alfalfa, nodule fresh weight and nitrogenase activity in Sinorhizobium meliloti strain S. 1021 meliloti model to study the mechanism of S. promoting effect of fulvic acid meliloti and nodulation nitrogen fixation. The results showed that fulvic acid significantly promoted the S. meliloti 1021 growth, the cell density increased by 5.07 times, and make it complete the quorum sensing system of S. strain meliloti 8530 (1021, expR+) the cell density was 20~50 times higher than that of fulvic acid. The relevant regulation and QS system. Real time fluorescence quantitative PCR (RT-qPCR) showed that fulvic acid significantly inhibited the quorum sensing related gene expR, 8530 S. meliloti sinlI, the expression of sinR and fulvic acid is mainly to regulate the quorum sensing system by inhibiting expression of expR gene. A comparative analysis of the mode of action of fulvic acid, fulvic acid could be found through the function of S. in meliloti 8530 QsrR protein. QsrR gene knockout confirmed the hypothesis in the meliloti Delta qsrR mutant, expR gene expression, and its expression is no longer inhibited by fulvic acid and gel retardation. The bacterial one hybrid experiment proved that QsrR protein can bind and inhibit the expression of expR gene and expR gene promoter and fulvic acid by increasing QsrR protein and expR gene promoter and further inhibit expR gene Expression. This is the first study to identify a regulated at the transcriptional level of expR gene expression protein QsrR, proved that the quorum sensing system of fulvic acid inhibition of S. meliloti8530 by QsrR and promote the growth of synthesis inhibition of EPS II and AHLs, in favor of rhizobia and weaken the host defense response in root colonization ring, which is conducive to symbiotic nitrogen fixation. The results in order to improve the efficiency of symbiotic nitrogen fixation has laid an important foundation. S. meliloti were obtained from 74 strains of rhizobia from Inner Mongolia Horqin Region. The matching experiment for eight high efficient Rhizobium alfalfa varieties in Horqin Region, primarily for each selection of alfalfa varieties to 1~3 strains of high efficient Rhizobium. The accumulation of excellent strains for alfalfa Rhizobium inoculation in Horqin sandy land area, but also enrich the alfalfa Rhizobium strain resources.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S541;S182
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本文编号：1724651