發(fā)布時間：2018-04-09 03:27

  本文選題：紫花苜蓿　切入點(diǎn)：苜蓿中華根瘤菌　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：苜蓿是一種具有很高經(jīng)濟(jì)價(jià)值和營養(yǎng)價(jià)值的多年生豆科牧草,目前苜蓿產(chǎn)量已經(jīng)嚴(yán)重供不應(yīng)求,限制了我國畜牧業(yè)發(fā)展。接種與苜蓿品種相匹配的高效根瘤菌不僅可以提高苜蓿產(chǎn)量和品質(zhì),而且可以減少氮肥使用,提高土壤肥力。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn)微生物降解褐煤產(chǎn)生的黃腐酸對多種禾本科作物都有很好的增產(chǎn)效果,而且能夠促進(jìn)大豆結(jié)瘤固氮,顯著提高大豆產(chǎn)量。本課題研究了兩株苜蓿高效根瘤菌Sinorhizibium meliloti CCBAU01199和Sinorhizibium meliloti CCBAU01290與中苜一號的共生有效性、黃腐酸對苜蓿產(chǎn)量的影響、黃腐酸對苜�！�根瘤菌體系的刺激作用及其機(jī)理,并針對內(nèi)蒙地區(qū)的八個苜蓿品種進(jìn)行了根瘤菌的分離及高效菌株的篩選,為提高苜蓿—根瘤菌體系的固氮效率,大力推廣苜蓿種植中接種根瘤菌提供理論和材料基礎(chǔ)。田間試驗(yàn)結(jié)果表明,接種兩株根瘤菌均能夠顯著提高苜蓿產(chǎn)量,而且S. meliloti CCBAU01199的增產(chǎn)效果高于S. meliloti CCBAU01290,說明S. meliloti CCBAU01199與中苜一號的匹配性更好。根瘤菌不同的接種方式也顯著影響接種效果,S. meliloti CCBAU01199最佳的接種方式是拌種,連續(xù)兩年使苜蓿產(chǎn)量分別提高了37.57%和24.78%。對土壤理化性質(zhì)測定結(jié)果表明,苜蓿接種根瘤菌提高了土壤全氮、堿解氮及有機(jī)質(zhì)含量,但使土壤有效磷和速效鉀含量降低,所以在苜蓿種植過程中需補(bǔ)施磷肥和鉀肥。噴施0.5g/L黃腐酸對苜蓿的增產(chǎn)效果與接種高效根瘤菌相似,連續(xù)兩年使苜蓿產(chǎn)量分別提高了29.57%和20.34%,接種S. meliloti CCBAU01290并噴施黃腐酸比只接種根瘤菌苜蓿增產(chǎn)9.16%。檢測苜蓿結(jié)瘤情況發(fā)現(xiàn)黃腐酸顯著提高了土著根瘤菌及接種根瘤菌與苜蓿的結(jié)瘤數(shù)、根瘤鮮重及固氮酶活。以苜蓿中華根瘤菌模式菌株S. meliloti 1021為對象研究了黃腐酸促進(jìn)S. meliloti與苜蓿結(jié)瘤固氮的作用機(jī)理。結(jié)果發(fā)現(xiàn),黃腐酸顯著促進(jìn)了S. meliloti 1021的生長,使其細(xì)胞密度提高了5.07倍,而且使具有完整群體感應(yīng)系統(tǒng)的菌株S. meliloti 8530 (1021,expR+)的細(xì)胞密度提高了20～50倍,說明黃腐酸的調(diào)控作用和群體感應(yīng)系統(tǒng)有關(guān)。實(shí)時熒光定量PCR(RT-qPCR)的結(jié)果顯示黃腐酸顯著抑制了S. meliloti 8530的群體感應(yīng)相關(guān)基因expR、sinlI、sinR的表達(dá),而且黃腐酸主要是通過抑制expR基因的表達(dá)而調(diào)控其群體感應(yīng)系統(tǒng)。對黃腐酸的作用模式進(jìn)行比較分析,發(fā)現(xiàn)黃腐酸可能是通過S. meliloti 8530中的QsrR蛋白起作用。對qsrR基因進(jìn)行敲除證實(shí)了上述假設(shè),在meliloti △qsrR突變株中,expR基因的表達(dá)上調(diào),而且其表達(dá)不再受黃腐酸的抑制。凝膠阻滯及細(xì)菌單雜交實(shí)驗(yàn)證明QsrR蛋白能夠與expR基因啟動子結(jié)合而直接抑制expR基因的表達(dá)。而且黃腐酸通過加強(qiáng)QsrR蛋白與expR基因啟動子的結(jié)合而進(jìn)一步抑制expR基因的表達(dá)。本研究首次鑒定了一個在轉(zhuǎn)錄水平調(diào)控expR基因表達(dá)的蛋白—QsrR,證明了黃腐酸通過QsrR抑制S. meliloti8530的群體感應(yīng)系統(tǒng)而促進(jìn)其生長,抑制其EPSⅡ及AHLs的合成,有利于根瘤菌在根圈的定殖并減弱宿主的防御反應(yīng),從而有利于共生固氮。研究結(jié)果為提高共生固氮效率奠定了重要基礎(chǔ)。從內(nèi)蒙科爾沁地區(qū)采集根瘤共分離得到S. meliloti 74株。針對科爾沁地區(qū)的八個苜蓿品種進(jìn)行高效根瘤菌的匹配實(shí)驗(yàn),初步為每個苜蓿品種篩選到1～3株高效根瘤菌。為科爾沁沙地地區(qū)的苜蓿接種根瘤菌積累了優(yōu)良菌株,也豐富了苜蓿高效根瘤菌菌種資源。
[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.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S541;S182
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本文編號：1724651