取样策略对SSR标记鉴别紫云英品种能力的影响

紫云英属于异花授粉植物, 品种内个体基因型杂合, 品种鉴定难度大。本文以紫云英闽紫系列3个审定品种为材料, 采用SSR标记进行取样策略对3个品种鉴别能力影响的研究。结果表明：（1）固定4对引物组合,从5～50进行梯度取样时,品种内的扩增位点总数、观测等位基因数趋于增多,但有效等位基因数、Shanon信息指数、遗传多样性指数增大到最大值后趋于下降,其中总体样品的最大值是出现在取样量为30的时候;随着样品量的增加,品种间Nei氏遗传距离以及分子方差分析的品种间期望变异系数比例值（PhiPT）均呈减少趋势,但PhiPT值的置信度在增大;（2）固定品种的样品容量为30和50,再加入2对能扩增出在品种间形成频率差异的标记位点的SSR引物对,基于这6对SSR引物可以将参试的3个紫云英品种有效的区分,品种间期望变异系数比例值（PhiPT）提高且差异的置信度具有明显的统计学意义（P<0.001）。主成分分析进一步表明：3个参试品种30个样品与50个样品的散布状况基本一致。对紫云英取样策略的研究表明：为提高对参试品种的鉴别能力,样品取样量以30株为宜,即达到较佳鉴别效果又降低分析成本。     

紫云英ISSR引物的筛选及PCR反应体系的优化

以紫云英为研究材料,用哥伦比亚大学(UBC)公布的100条ISSR引物和11种株系紫云英品种的DNA为模板进行PCR扩增,筛选出33条扩增条带较好的ISSR引物,对其中的ISSR引物进行梯度PCR,筛选出最佳的退火温度。再采用正交试验和单因素试验相结合的方法对紫云英ISSR-PCR反应体系的5种因素(模板、Mg2+、TaqDNA聚合酶、dNTP及引物)进行优化浓度。确立了适合紫云英的ISSR分析的反应体系。在25μl反应体系中,其反应浓度为:DNA模版50.00ng,Mg2+2.00mol/L,Taq聚合酶1.0 U,dNTP 0.25mmol/L,引物0.20μmol/L,2.5μl 10×buffer。本试验为以后利用ISSR技术进行紫云英遗传多样性分析和物种保护奠定了技术基础。     

紫云英AD-cDNA文库构建及与豆血红蛋白Lb相互作用靶蛋白的筛选

【目的】本文旨在构建紫云英酵母双杂交AD-cDNA文库和互作靶蛋白筛选平台,为深入研究共生固氮作用的分子机理奠定工作基础。【方法】以接种华癸中慢生根瘤菌7653R的豆科植物紫云英不同时期根部组织为材料,抽提和纯化RNA,构建了一个酵母AD-cDNA文库。库容量达到1.02×106/3μg pGADT7-RecDNA,插入片段大小1-1.5 kb左右。以紫云英豆血红蛋白基因AsB2510构建诱饵载体pGBKT7-AsB2510,利用酵母双杂交技术,筛选与诱饵蛋白相互作用的靶蛋白。【结果】在含有X-gal的SD四缺培养基上筛选得到26个克隆,经过质粒抽提、PCR鉴定、回转酵母验证获得10个阳性克隆。【结论】对阳性克隆的外源片段进行了测序和同源性分析,发现一个值得深入研究的含有tify domain和Divergent CCT motif的转录调控因子。     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.     

紫云英配施化肥对水稻氮素吸收利用和紫云英氮在水稻-土壤体系分配、残留的影响

明确紫云英配施化肥条件下水稻对氮素吸收利用和紫云英氮在水稻-土壤体系的吸收利用、分配及残留规律,能够为豫南稻区合理施肥提供依据.本研究利用原状土柱模拟和15N示踪技术,研究等氮条件下不施肥(CK)、化肥+22500 kg·hm-2紫云英(FM1)、化肥+30000 kg·hm-2紫云英(FM2)、化肥+37500 kg...     

一株高效降解芘的细菌分离、鉴定及其降解效果

摘要：【目的】获得高效降解高分子量多环芳烃的细菌,并研究其对多环芳烃的降解能力。【方法】利用富集培养和芘升华平板方法,从焦化厂污染土壤中分离多环芳烃降解细菌,对分离菌株通过形态特征、16S rRNA基因和gyrb基因序列相似性分析进行鉴定,并研究该菌对高分子量多环芳烃（HMW-PAHs）的降解效果。【结果】筛选到一株能以芘、苯并蒽、屈、苯并芘、茚并芘、苯并苝、荧恩为碳源和能源生长并降解这些底物的菌株HBS1,该菌株的16S rRNA基因和gyrb基因序列与Gordonia amicalis的相应基因的相似     

分枝杆菌TZh51菌株的分离鉴定及其生物修复污染土壤的特性

[目的]为获得降解芘的微生物菌株,并用其生物修复被多环芳烃污染的土壤.[方法]芘降解菌的分离采用平板升华法.根据表型观察、生理生化特性和16S rDNA的序列同源性分析,对菌株进行分类学鉴定.通过活菌计数、HPLC测定多环芳烃的残留量,研究菌株在固体、液体无机盐培养基以及在污染土壤中降解多环芳烃(polycyclic aromatic hydrocarbons,PAHs)的能力.[结果]分离到4株能降解芘的菌株TZh51、TZh52、TG42和TG52.实验结果表明,TZh51降解PAHs的能力强于其余3株菌.TZh51被鉴定为分枝杆菌属(Mycobacterium sp.),但与已发表的分枝杆菌菌株M11为不同的种.TZh51接种在芘膜的固体无机盐培养基上,测定获得最大芘降解量的条件是培养温度为3512和芘膜厚度为130 ng/mm2.在芘浓度为50、100 mg/L的液体无机盐培养基中培养,6天时TZh51的芘降解率分别达到91.9%、71.8%,10天时菌体数量分别达到最大值为2.0、6.0×108cfu/mL;TZh51降解芘的效果强于M11.在种植作物的处理中,到第6周时TZh51的菌体数量达到每克干土含7.2×108个菌落数,到第8周时菲、荧蒽和芘的降解率分别达到91.4%、86.9%和85.8%;[结论]TZh51具有很强降解PAHs的能力;另外,TZh51与作物联合生物修复污染土壤的效果明显.     

Isolation and characterization of a biosurfactant‐producing Fusarium sp. BS‐8 from oil contaminated soil

This study reports characterization of a biosurfactant‐producing fungal isolate from oil contaminated soil of Missa Keswal oil field, Pakistan. It was identified as Fusarium sp. BS‐8 on the basis of macroscopic and microscopic morphology, and 18S rDNA gene sequence homology. The biosurfactant‐producing capability of the fungal isolates was screened using oil displacement activity, emulsification index assay, and surface tension (SFT) measurement. The optimization of operational parameters and culture conditions resulted in maximum biosurfactant production using 9% (v/v) inoculum at 30°C, pH 7.0, using sucrose and yeast extract, as carbon and nitrogen sources, respectively. A C:N ratio of 0.9:0.1 (w/w) was found to be optimum for growth and biosurfactant production. At optimal conditions, it attained lowest SFT (i.e., 32 mN m^?1) with a critical micelle concentration of ≥ 1.2 mg mL^?1. During 5 L shake flask fermentation experiments, the biosurfactant productivity was 1.21 g L^?1 pure biosurfactant having significant emulsifying index (E₂₄, 70%) and oil‐displacing activity (16 mm). Thin layer chromatography and Fourier transform infrared spectrometric analyses indicated a lipopeptide type of the biosurfactant. The Fusarium sp. BS‐8 has substantial potential of biosurfactant production, yet it needs to be fully characterized with possibility of relatively new class of biosurfactants. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1065–1075, 2014     

Isolation of biosurfactant producers, optimization and properties of biosurfactant produced by Acinetobacter sp. from petroleum-contaminated soil

Aims: To screen and identify biosurfactant producers from petroleum‐contaminated soil; to use response surface methodology (RSM) for medium optimization to enhance biosurfactant production; and to study the properties of the newly obtained biosurfactant towards pH, temperature and salinity. Methods and Results: We successfully isolated three biosurfactant producers from petroleum‐contaminated soil and identified them through 16S rRNA sequence analysis, which exhibit the highest similarities to Acinetobacter beijerinckii (100%), Kocuria marina (99%) and Kineococcus marinus (99%), respectively. A quadratic response model was constructed through RSM designs, leading to a 57·5% increase of the growth‐associated biosurfactant production by Acinetobacter sp. YC‐X 2 with an optimized medium: beef extract 3·12 g l^?1; peptone 20·87 g l^?1; NaCl 1·04 g l^?1; and n‐hexadecane 1·86 g l^?1. Biosurfactant produced by Acinetobacter sp. YC‐X 2 retained its properties during exposure to a wide range of pH values (5–11), high temperatures (up to 121°C) and high salinities [up to 18% (w/v) Na⁺ and Ca²⁺], which was more sensitive to Ca²⁺ than Na⁺. Conclusions: Two novel biosurfactant producers were isolated from petroleum‐contaminated soil. Biosurfactant from Acinetobacter sp. YC‐X 2 has good properties to a wide range of pH, high temperature and high salinity, and its production was optimized successfully through RSM. Significance and Impact of the Study: The fact, an increasing demand of high‐quality surfactants and the lack of cost‐competitive bioprocesses of biosurfactants for commercial utilization, motivates researchers to develop cost‐effective strategies for biosurfactant production through isolating new biosurfactant producers with special surface‐active properties and optimizing their cultural conditions. Two novel biosurfactant producers in this study will widen our knowledge about this kind of micro‐organism. This work is the first application of RSM designs for cultural optimization of biosurfactant produced by Acinetobacter genus and the first report that biosurfactant may be more sensitive to Ca²⁺ than Na⁺.     

象白蚁肠道中一个纤维素降解菌群的分离和特性研究

利用滤纸培养基从象白蚁(Nasutitermes sp.)肠道中分离出一个具有纤维素降解能力,能够降解滤纸的混合菌群。在起始pH 6.5,37℃培养条件下培养6d可得到最高的纤维素酶(CMCase和FPase)活性。在优化条件下,混合菌群的滤纸降解率在第15d达到最大值66.3%,显示出较高的滤纸降解效率。酶谱活性染色分析显示,混合菌群在以滤纸为唯一碳源的生长过程中至少表达了8种内切葡聚糖酶和4种木聚糖酶。扫描电镜观察到该混合菌群包含短杆状和球形两种形态的细菌。基于16SrRNA基因的系统发育分析表明,该混合菌群中至少存在两种细菌,分别属于沙雷氏菌属(Serratia)和类芽胞杆菌属(Paenibacillus)。这两种细菌协同降解纤维素的机制值得进一步深入研究。     

Characterization of Rhizobium sp. Q32 Isolated from Weathered Rocks and its Role in Silicate Mineral Weathering

One mineral-solubilizing strain was isolated from weathered potassic trachyte surfaces and identified as Rhizobium sp. Q32 based on the 16S rRNA gene sequence analysis. The mineral (feldspar and biotite) dissolution potential and the physiological characteristics of the bacterium were investigated. Laboratory mineral dissolution experiments indicated that inoculation with the live bacterium significantly increased feldspar and biotite dissolution by a factor of 1.2–4.7 for Si and 1.2–1.5 for K in comparison with the dead bacterium inoculated controls. In addition, extracellular polysaccharide production by the bacterium increased with time but the bacterium produced small pH changes (6.0–6.5) in the course of mineral dissolution experiment. The bacterium was found to produce siderophores and have the characteristics of acid or alkali and salt tolerance and temperature resistance. The result suggested that feldspar and biotite dissolution may be mainly caused by extracellular polysaccharide and/or siderophores produced by the bacterium.     

石灰性土壤拉恩式溶磷细菌的筛选鉴定及溶磷特性

从山西石灰性土壤作物根际分离筛选出多株溶磷细菌,经过多次分离纯化得到一株溶磷能力较强的菌株W25,通过菌落形态、生理生化特性和16S rRNA序列分析,确定溶磷菌W25为拉恩式菌属.对W25溶解磷特性进一步研究表明:其对磷酸三钙、磷酸铝和磷酸铁最高溶磷能力分别为385.5、110.4、216.6 mg·L-1;在磷酸铝和磷酸铁培养液中,W25溶磷量与培养液pH的相关系数分别为0.56和0.81,呈极显著负相关;在不同碳氮源条件下,W25以葡萄糖为碳源和NH4NO3为氮源时对磷酸三钙的溶磷量最高,对碳源的利用顺序依次为葡萄糖＞乳糖＞蔗糖＞甘露醇＞淀粉,对氮源的利用顺序依次为NH4NO3 ＞NH4Cl＞(NH4)2SO4＞NaNO3＞KNO3.不同氮源对W25产生有机酸的种类影响较大,以铵态氮为氮源产生甲酸和乙酸,以硝态氮为氮源产生草酸和琥珀酸,以硝酸铵为氮源还产生柠檬酸.     

湖榕和小叶榕花形态结构特征的研究

通过对湖榕（Ｆｉｃｕｓ　ｓｐ．）和小叶榕（Ｆｉｃｕｓ　ｍｉｃｒｏｃａｒｐａ　Ｌ．）花形态结构特征的比较研究表明：湖榕和小叶榕的花果形态结构有较显著的差别。湖榕雌花的萼片数多为４片,萼片顶部都有毛,花粉位为精园形,花粉壁较薄,其结实率较低,只有０．１％;小叶榕雌花的萼片数多为３片,萼片顶部光滑无毛,花粉粒为圆形而壁较厚,其结实率较高,为２９．１％。     

一株可利用甲烷的克雷伯氏菌的分离及其在甲烷检测中的应用

从山西太原水稻田土壤中,分离得到一株能以甲烷为唯一碳源和能源生长的菌株C611。通过生理生化特征及16SrDNA序列分析,该菌株初步鉴定为克雷伯氏菌(Klebsiella sp.)。采用响应面法优化了该菌株利用甲烷的培养条件,得到最佳培养条件为:温度24.4oC、接种量为6.7%、甲烷含量25%。以C611固定化细菌和溶氧响应仪为体系,采用电化学法研究了不同含量甲烷的响应时间以及溶氧变化与甲烷含量的关系。结果表明,菌株C611能利用甲烷,该反应体系对0～10%甲烷气体测定的响应时间小于100s;溶氧消耗量与通入甲烷气体含量呈线性关系,拟合系数(R2)为0.9994。以3%甲烷气体样品进行8次测量,测定平均值为3.09%,RSD为3.48%,相对误差为3%。表明该反应体系重现性良好,为该菌株进一步研究甲烷传感器奠定基础。     

Mesorhizobium jarvisii is a dominant and widespread species symbiotically efficient on Astragalus sinicus L. in the Southwest of China

In order to identify rhizobia of Astragalus sinicus L. and estimate their geographic distribution in the Southwest China, native rhizobia nodulating A. sinicus were isolated and their genetic diversity were studied at 13 sites cultivated in four Chinese provinces. A total of 451 rhizobial isolates were trapped with A. sinicus plants from soils and classified into 8 different genotypes defined by PCR-based restriction fragment length polymorphism (RFLP) of 16S–23S rRNA intergenic spacer (IGS). Twenty-one representative strains were further identified into three defined Mesorhizobium species by phylogenetic analyses of 16S rRNA genes and housekeeping genes (glnII and atpD). M. jarvisii was dominant accounting for 76.3% of the total isolates, 22.8% of the isolates were identified as M. huakuii and five strains belonged to M. qingshengii. All representatives were assigned to the symbiovar astragali by sharing high nodC sequence similarities of more than 99%. Furthermore, the biogeography distribution of these rhizobial genotypes and species was mainly affected by contents of available phosphorus, available potassium, total salts and pH in soils. The most remarkable point was the identification of M. jarvisii as a widespread and predominant species of A. sinicus in southwest of China. These results revealed a novel geographic pattern of rhizobia associated with A. sinicus in China.     

根瘤菌N613胞外多糖发酵条件及抗肿瘤作用研究

用摇瓶正交试验研究了根瘤菌Rhizobium sp.N613合成根瘤菌胞外多糖(rhizobium exopolysaccharide,REPS)的最佳培养基配方和最适培养条件。采用10L自动控制罐进行了分批发酵试验,获得了合成REPS的发酵动力学参数。经40h的补料分批发酵与代谢调控,REPS产量达到11.31g/L。此外,抗肿瘤实验表明,当REPS剂量为5mg/kg时,其抑瘤率可达53.40%。结果表明,REPS的发酵周期短,产量高,成本低,且具有良好的免疫活性与增稠性,有着极高的开发应用价值。     

Gordonia sp. LAM0048的分离鉴定及其降解正十六烷的研究

以正十六烷为唯一碳源,从长期受石油污染的土壤中筛选到一株高效降解正十六烷的菌株LAM0048。通过形态学观察、生理生化试验、细胞化学组分分析、16S rRNA基因序列分析、细胞脂肪酸和极性脂试验,确定其属于棒杆菌亚目(Corynebacterineae)、诺卡菌科(Nocardiaceae)、戈登氏属(Gordonia),且可能为戈登氏属的一株新种。采用单因素实验对菌株LAM0048在无机盐培养基中降解正十六烷的降解率进行初步探讨,发现该菌株能在以正十六烷为唯一碳源的培养基中生长,菌株LAM0048能够在36 h内完全降解0.05%(V/V)的正十六烷,当烷烃浓度达到1.0%(V/V)时,降解率达46.4%。结果表明LAM0048是一株具有耐受高浓度烷烃的石油降解菌,在石油污染环境的微生物修复中具有一定的应用潜力。     

Mycobacterium fluoranthenivorans sp. nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant

Mycobacterium strain FA4^T was isolated with fluoranthene as the single carbon source from soil of a former coal gas plant, polluted with polycyclic aromatic hydrocarbons. The physiological properties, fatty acid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobacterium, but were different from all type strains of Mycobacterium species. Based on comparative 16S rRNA gene sequence analyses strain FA4^T could be assigned to the Mycobacterium neoaurum taxon showing 98% sequence similarity to M. diernhoferi as its closest neighbour. The occurrence of epoxymycolate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester mycolates in addition to alpha-mycolates. Strain FA4^T is able to degrade aflatoxin B₁. This biological attribute might be useful in biological detoxification processes of foods and feeds. From the investigated characteristics it is concluded that strain FA4^T represents a new species, for which we propose the name Mycobacterium fluoranthenivorans sp. nov. The type strain of Mycobacterium fluoranthenivorans is FA4^T (DSM 44556^T = CIP 108203^T).