固氮内生菌HMLR8和HMLR13对水稻的促生效应的研究
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摘要
通过表面消毒,组织碾碎,汁液涂布无氮培养基的方法从稗草根中分离到内生固氮菌18株,茎中分离到内生固氮菌17株。
经回接水稻实验后,通过形态学和分子鉴定技术比较筛选得到两株能侵染水稻中花11同时促进水稻生长的菌株HMLR8和HMLR13。通过生理生化实验及16SrDNA测序比较得出它们分别为Klebsiella oxytoca和Roseateles depolymerans。PCR方法扩增nifH基因和乙炔还原法测定固氮酶活性证明了HMLR8和HMLR13的固氮性质。用改良的Do氏培养基培养HMLR8和HMLR13,测到它们的固氮酶活性分别为1721.11nmolC2H4·ml-1·h-1和204.68 nmolCH4·ml-1·h-1。通过测定不同碳源条件下HMLR13的固氮酶活性发现HMLR13在以甘油为唯一碳源时固氮酶活性最高,达到406.5593 nmolC2H4ml-1h-1。
利用Roseateles depolymeran光捕捉器操纵子上的一段特异序列ORF077,为HMLR13设计了一对种特异性引物,并初步验证了其特异性,同时用接种HMLR13的水稻组织提取细菌DNA扩增得到正确的特异条带,为检测Roseateles depolymerans strain HMLR13在环境中的存在及宿主范围提供了一种更为简便的方法。
通过不同浓度药物培养基培养的梯度筛选方法,得到具有100μg/mL利福平抗性的HMLR8和HMLR13菌株,以此测定HMLR8和HMLR13在水稻组织内的定殖动态。研究结果表明在蘸根接种时,剪根和不剪根两种条件下,两株菌均能正常侵染水稻。其中HMLR13在水稻根内的定殖数量在第5天达到最大,约为6.0×106cfu/gFW, HMLR8在水稻根内的定殖数量在第10天时达到最大,为5.9×106cfu/gFW。这表明HMLR8在水稻根内的定殖能力强于HMLR13。实验还表明在接种3天后才在茎中检测到HMLR8和HMLR13的存在,并且它们的数量从接种后第3天到第14天一直呈上升趋势。
应用接合转移的方法对HMLR13进行gfp分子标记,限菌条件下回接无氮水稻培养基培养的水稻,激光共聚焦显微镜观察其在水稻根茎叶组织中的分布。实验结果表明HMLR8和HMLR13在水稻根,茎和叶的细胞内和细胞间隙都有分布,且HMLR13和HMLR8的纤维素水解酶和果胶水解酶活性协助了它们在水稻组织内的定殖和迁移。实验还表明当用HMLR8和HMLR13接种用无氮半固体水稻培养基培养的水稻时,它们在水稻根表有聚集附着现象,并随着根的生长向培养基内部移动。同时,接种了HMLR8和HMLR13的水稻的根毛要明显多于对照。测定接种了HMLR8和HMLR13的水稻植株的固氮酶活性,结果分别为0.359nmolC2H4-mgFW-1·h-1和0.760.359nmolC2H4·mgFW-1·h-1,这表明水稻组织内的微环境更适合HMLR8固氮酶活性的表达。
盆栽实验中,在水稻分别接种HMLR8和HMLR13后60天收获植物分别测定植株的鲜重,干重,根长,株高和植株总氮含量等数据。实验结果表明HMLR8和HMLR13均能不同程度地提高水稻生物量、根长、芽长和植株氮含量。HMLR8根长比对照高出115.58%,株高比对照高出47.69%,鲜重比对照高出140.14%,干重比对照高出162.91%,植株含氮量比对照高出241.87%。HMLR13根长比对照高出95.86%,株高比对照高出27.39%,鲜重比对照高出61.47%,干重比对照高出63.04%,植株含氮量比对照高出43.42%。这表明HMLR8对水稻的生长促进作用要大于HMLR13。
35 endophytic diazotrophic bacteria from surface-sterilized roots and stems of Echinochloa crusgalli which grows luxuriantly with no additional nitrogen fertilizer were isolated. Of all these bacteria, two isolates HMLR8 and HMLR13 showed the ability of infecting rice variety ZH-11 seedlings and growth-promoting property. These isolates were identified as Klebsiella oxytoca strain HMLR8 and Roseateles depolymerans strain HMLR13 respectively on the basis of 16S rDNA sequence and biochemical tests analysis. Amplification of nifH by polymerase chain reaction (PCR) and detection of nitrogenase activity by acetylene reduction assay confirmed the diazotrophic nature of HMLR8 and HMLR13. As an endophytic diazotrophic bacterium never been reported, HMLR13 was tagged with gfp gene and the resulting conjugant was inoculated to endophyte-free rice seedlings to detect its colonization and distribution in the tissues of rice seedlings. The results demonstrated that:(1) incubation of these two isolates especially HMLR8 resulted in significant increase in root/shoot length and nitrogen content of rice seedlings; (2) Roseateles depolymerans strain HMLR13 was a new endophytic diazotrophic bacterium which displays maximum nitrogenase activity of 406.5593 nmol C2H4ml-1h-1 when using glycerine as single carbon source.
经回接水稻实验后,通过形态学和分子鉴定技术比较筛选得到两株能侵染水稻中花11同时促进水稻生长的菌株HMLR8和HMLR13。通过生理生化实验及16SrDNA测序比较得出它们分别为Klebsiella oxytoca和Roseateles depolymerans。PCR方法扩增nifH基因和乙炔还原法测定固氮酶活性证明了HMLR8和HMLR13的固氮性质。用改良的Do氏培养基培养HMLR8和HMLR13,测到它们的固氮酶活性分别为1721.11nmolC2H4·ml-1·h-1和204.68 nmolCH4·ml-1·h-1。通过测定不同碳源条件下HMLR13的固氮酶活性发现HMLR13在以甘油为唯一碳源时固氮酶活性最高,达到406.5593 nmolC2H4ml-1h-1。
利用Roseateles depolymeran光捕捉器操纵子上的一段特异序列ORF077,为HMLR13设计了一对种特异性引物,并初步验证了其特异性,同时用接种HMLR13的水稻组织提取细菌DNA扩增得到正确的特异条带,为检测Roseateles depolymerans strain HMLR13在环境中的存在及宿主范围提供了一种更为简便的方法。
通过不同浓度药物培养基培养的梯度筛选方法,得到具有100μg/mL利福平抗性的HMLR8和HMLR13菌株,以此测定HMLR8和HMLR13在水稻组织内的定殖动态。研究结果表明在蘸根接种时,剪根和不剪根两种条件下,两株菌均能正常侵染水稻。其中HMLR13在水稻根内的定殖数量在第5天达到最大,约为6.0×106cfu/gFW, HMLR8在水稻根内的定殖数量在第10天时达到最大,为5.9×106cfu/gFW。这表明HMLR8在水稻根内的定殖能力强于HMLR13。实验还表明在接种3天后才在茎中检测到HMLR8和HMLR13的存在,并且它们的数量从接种后第3天到第14天一直呈上升趋势。
应用接合转移的方法对HMLR13进行gfp分子标记,限菌条件下回接无氮水稻培养基培养的水稻,激光共聚焦显微镜观察其在水稻根茎叶组织中的分布。实验结果表明HMLR8和HMLR13在水稻根,茎和叶的细胞内和细胞间隙都有分布,且HMLR13和HMLR8的纤维素水解酶和果胶水解酶活性协助了它们在水稻组织内的定殖和迁移。实验还表明当用HMLR8和HMLR13接种用无氮半固体水稻培养基培养的水稻时,它们在水稻根表有聚集附着现象,并随着根的生长向培养基内部移动。同时,接种了HMLR8和HMLR13的水稻的根毛要明显多于对照。测定接种了HMLR8和HMLR13的水稻植株的固氮酶活性,结果分别为0.359nmolC2H4-mgFW-1·h-1和0.760.359nmolC2H4·mgFW-1·h-1,这表明水稻组织内的微环境更适合HMLR8固氮酶活性的表达。
盆栽实验中,在水稻分别接种HMLR8和HMLR13后60天收获植物分别测定植株的鲜重,干重,根长,株高和植株总氮含量等数据。实验结果表明HMLR8和HMLR13均能不同程度地提高水稻生物量、根长、芽长和植株氮含量。HMLR8根长比对照高出115.58%,株高比对照高出47.69%,鲜重比对照高出140.14%,干重比对照高出162.91%,植株含氮量比对照高出241.87%。HMLR13根长比对照高出95.86%,株高比对照高出27.39%,鲜重比对照高出61.47%,干重比对照高出63.04%,植株含氮量比对照高出43.42%。这表明HMLR8对水稻的生长促进作用要大于HMLR13。
35 endophytic diazotrophic bacteria from surface-sterilized roots and stems of Echinochloa crusgalli which grows luxuriantly with no additional nitrogen fertilizer were isolated. Of all these bacteria, two isolates HMLR8 and HMLR13 showed the ability of infecting rice variety ZH-11 seedlings and growth-promoting property. These isolates were identified as Klebsiella oxytoca strain HMLR8 and Roseateles depolymerans strain HMLR13 respectively on the basis of 16S rDNA sequence and biochemical tests analysis. Amplification of nifH by polymerase chain reaction (PCR) and detection of nitrogenase activity by acetylene reduction assay confirmed the diazotrophic nature of HMLR8 and HMLR13. As an endophytic diazotrophic bacterium never been reported, HMLR13 was tagged with gfp gene and the resulting conjugant was inoculated to endophyte-free rice seedlings to detect its colonization and distribution in the tissues of rice seedlings. The results demonstrated that:(1) incubation of these two isolates especially HMLR8 resulted in significant increase in root/shoot length and nitrogen content of rice seedlings; (2) Roseateles depolymerans strain HMLR13 was a new endophytic diazotrophic bacterium which displays maximum nitrogenase activity of 406.5593 nmol C2H4ml-1h-1 when using glycerine as single carbon source.
引文
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