奥利亚罗非鱼和尼罗罗非鱼SRY基因同源克隆和序列分析

采用人SRY基因保守序列的特异性引物，对奥利亚罗非鱼（Orcocbromis aureus）和尼罗罗非鱼（O．nilotica）雌、雄个体基因组进行PCR扩增，回收其中的600bp片段，分离克隆并双向测序。用DNAStar分析比较所得序列，表明克隆的600bp片段为SRY基因的同源序列。对两种罗非鱼雌、雄个体SRY同源序列进行多序列比较分析（CLUSTAL W分析），表明罗非鱼间SRY同源序列的相似性非常高，但仍存在性别间、种间差异。奥利亚罗非鱼雌、雄间序列相似性为99．5％，尼罗罗非鱼雌、雄间为99．5％，奥利亚罗非鱼与尼罗罗非鱼种间则为98．7％，罗非鱼与人类SRY基因间只有7．1％，结果为进一步研究罗非鱼SRY同源基因与性别决定的关系提供了可能性。根据这些结果构建了罗非鱼系统分类树，表明奥利亚罗非鱼与尼罗罗非鱼具有很近的亲缘关系。     

三种罗非鱼的微卫星分子鉴定和遗传结构分析

采用77对微卫星引物对奥利亚、尼罗和橙色莫桑比克三种罗非鱼基因组DNA进行PCR扩增,筛选出17个微卫星鉴定位点,其中奥利亚罗非鱼的特异位点UNH636、UNH117、UNH172、UNH738、UNH878、UNH896;尼罗罗非鱼的特异位点UNH913、UNH907、UNH222、UNH980 、UNH880和橙色莫桑比克罗非鱼的特异位点UNH876、UNH899、UNH853、UNH932、UNH933、UNH773,任意一个位点可在其中一种罗非鱼中扩增出其它两种罗非鱼不具有的条带,从而将这种罗非鱼与其它两种区分开来。利用这17个位点检测三种罗非鱼的遗传结构,共检测到142个等位基因,平均等位基因8.35个,数据经Popgen32计算和MEGA4软件作图,进行了聚类分析,确立了三种罗非鱼的亲缘关系。结果表明,奥利亚、尼罗、橙色莫桑比克三种罗非鱼的平均观测杂合度分别为0.0941、0.5490、0.2588,平均期望杂合度分别为0.1089、0.7230、0.2608,平均多态信息含量分别为0.0869、0.7149 、0.1643,由此可见,尼罗罗非鱼群体的遗传多样性水平最高,奥利亚罗非鱼群体遗传多样性最低;聚类分析显示奥利亚群体和橙色莫桑比克群体间的亲缘关系较近,与前人研究结果一致。     

尼罗罗非鱼胰岛的显微和亚显微结构

应用光镜、电镜及特殊染色等技术对尼罗罗非鱼(Tilapia nilotica)的胰岛及其细胞类型和形态进行了详细的研究。尼罗罗非鱼的胰脏与肝脏混合存在，属于肝胰脏。胰岛散布在胰腺内，无固定的形态，胰岛细胞的数量与类型因胰岛的大小而异。用Gomori醛复红特殊染色法，见尼罗罗非鱼胰岛有三种细胞：A细胞胞质中有黄色颗粒；B细胞胞质中有紫红色的颗粒；D细胞胞质内有浅绿色或淡灰色的颗粒。电镜下，B细胞的分泌颗粒较大,芯的形态多样，电子密度差异大,芯与界膜之间常有较大的间隙；A细胞分泌颗粒形态不规则，芯的大小不等，界膜与芯之间无间隙；D细胞分泌颗粒较多，芯的电子密度较低，界膜与芯之间无间隙。此外，胞质内可见较大的线粒体。     

光合细菌PSB07—15对水培黄瓜体系中甲氰菊酯污染的生物修复

为了探索光合细菌PSB07—15降解甲氰菊酯的应用潜力,在实验室研究了光合细菌PSB07—15对水培黄瓜体系中的甲氰菊酯污染的生物修复效率。结果表明,培养30d,光合细菌PSB07—15对黄瓜营养液中100mg·L^-1甲氰菊酯降解率达到47．63％,黄瓜中甲氰菊酯降解率达59．73％。光合细菌PSB07—15可以使黄瓜的根长和生物量显著增加,而黄瓜的根活力以及根H2O2酶活力增加并不显著。     

光合细菌对罗非鱼塘中水质和生态的影响

研究了在罗非鱼池塘养殖过程中投以光合细菌后，对池塘水质及水中微生物种群数量的变化影响，通过2个试验组和2个对照组进行为期半个月的试验。试验结果表明：（1）投放光合细菌的池塘中，光合细菌种群由少到多不断地增长，并且形成优势种群；（2）投放光合细菌后，对降低水中COD、氨氮，提高亚硝酸态氮等有一定的效果；（3）但是对水中溶氧、pH值、硝酸态氮等的变化并没有起太大的作用。     

尼罗罗非鱼β2m基因的克隆、多态性分析及组织表达特征

β2-微球蛋白(β2-microglobulin,β2m)是主要组织相容性复合体(major histocompatibility complex,MHC)Ⅰ分子的亚基之一,与MHC-Ioα链非共价结合构成MHC Ⅰ分子.本实验通过反转录PCR(RT-PCR)和cDNA末端快速扩增技术(rapid-amplification of cDNA ends,RACE)克隆了尼罗罗非鱼(Oreochromis niloticus) β2m基因的全长cDNA序列及基因组序列,并对cDNA多态性、mRNA组织分布及感染链球菌后表达的变化进行了分析.结果共获得尼罗罗非鱼2条β2m基因cDNA,全长分别为900和906 bp.两cDNA均包括351 bp的ORF,共编码116个氨基酸残基,还包含68 bp的5'非编码区(5'-UTR)和486(492) bp的3'-UTR.两条cDNA对应各自不同的基因组序列.基因组序列分析发现,β2m基因含3个外显子和2个内含子.尼罗罗非鱼β2m基因推测氨基酸序列与其他物种的β2m基因相似性在39.20％～89.80％之间.β2m基因cDNA多态性分析表明,尼罗罗非鱼中共有2种不同类型的β2m cDNA,每种类型cDNA又有3种不同的亚型.定量PCR分析表明,β2m基因在尼罗罗非鱼脾脏、心脏和肾脏中表达量最高,鳃与肠中的表达量较高,在皮肤和肌肉中的表达量最低.腹腔注射无乳链球菌(Streptococcus agalactiae)后,尼罗罗非鱼β2mmRNA表达量在4个所检测组织(鳃,肾脏,心脏和脾脏)中均出现了先上升后下降的变化趋势.本实验结果表明,β2m基因可能参与尼罗罗非鱼的免疫应答,在免疫反应中发挥重要功能.本研究有助于进一步了解尼罗罗非鱼的免疫调节机制和更好地理解鱼β2类m的生理功能.     

两个罗非鱼杂交组合亲本群体与子一代的AFLP分析

运用AFLP指纹技术分别对尼罗罗非鱼和奥利亚罗非鱼及它们的杂交子一代、橙色莫桑比克罗非鱼和荷那龙罗非鱼及它们的杂交子一代的遗传结构进行分析。从90对选扩引物组合中筛选出8对进行PCR扩增，8对引物组合共检出250个扩增位点（片段大小100～500bp）。每一个体的每对引物扩增条带数在20～47之间。群体内遗传相似度从高到低依次为：奥利亚罗非鱼（0.913），尼罗罗非鱼（0.871），荷那龙罗非鱼（0.829）和橙色莫桑比克罗非鱼（0.784）。奥利亚罗非鱼和尼罗罗非鱼间的遗传相似度是0.811，遗传距离为0.189；奥利亚罗非鱼和尼罗罗非鱼与杂交子一代的遗传距离分别为0.165、0.142。橙色莫桑比克罗非鱼和荷那龙罗非鱼间的遗传相似度0.756，遗传距离为0.244；橙色莫桑比克罗非鱼和荷那龙罗非鱼与杂交子一代的遗传距离分别为 0.148、0.162。引物组合E-AGG/M-CTT在尼罗罗非鱼、E-ACA/M-CAC在尼奥鱼中扩增出差异的DNA片段，可作为区分奥利亚罗非鱼和尼罗罗非鱼与杂交子一代的分子标记。     

外源生长激素对尼罗罗非鱼生长、骨骼肌纤维增生及肥大的影响

生长激素是影响动物生长发育的最重要内分泌激素,为了探讨外源生长激素对尼罗罗非鱼(Oreochromis niloticus)生长、骨骼肌纤维增生以及肥大的影响,本实验对同一批次尼罗罗非鱼幼鱼进行为期7周的外源重组人生长激素(hGH)肌肉注射,并设置了磷酸盐缓冲液(PBS)注射对照组,每周注射1次,测量体重、体长等表型生长指标,同时,隔两周采集背部肌肉,利用石蜡切片技术分析骨骼肌背侧第一肌节中肌纤维数目与面积的变化.结果显示:(1)注射后第1周起,实验组鱼的体重、体长即高于对照组,但差异不显著(P＞0.05);第5周起,实验组鱼体重显著高于对照组鱼(P＜0.05),第7周起,实验组鱼体长极显著高于对照组鱼(P＜0.01).(2)注射后第1周起,实验组鱼肌纤维数目、总面积均高于对照组鱼,差异不显著(P＞0.05);第5周起,实验组鱼肌纤维的外源性增生和肥大现象极显著(P＜0.01).研究结果表明,外源生长激素对尼罗罗非鱼的表型生长、骨骼肌肌纤维的增生与肥大均有显著促进作用,肌肉生长中以肥大生长更为明显.     

甲萘威暴露对雄性罗非鱼血清中内分泌相关酶活性的影响

实验室条件下,尼罗罗非鱼暴露于不同浓度的甲萘威（0．16、0．8、2mg·L^-1）下29d,通过酶联免疫法测定黄体生成素（LH）、卵泡刺激素（FSH）、三碘甲状腺原氨酸（T3）、甲状腺素（T4）水平变化,探讨甲萘威对尼罗罗非鱼内分泌干扰效应。研究结果显示,在实验期间与对照组相比,0．16、0．8、2mg·L^-1甲萘威暴露下的LH、FSH、T3、T4水平均随暴露时间增加不断升高,其中：10-19d内上升幅度小,20～29d内上升幅度较大,在实验第29d,LH、FSH、T3、T4水平均达到暴露以来的最大值。研究表明,从甲萘威对鱼体存在10d的短期积累效应来看,O．8mg·L^-1的甲萘威具有最强的内分泌干扰能力,此浓度下的LH、FSH、T3、T4水平在暴露第10d均高于其他两个浓度组;从29d的长期积累效应来看,2mg·L^-1浓度组的各激素水平上升幅度最大。     

广东养殖尼罗罗非鱼3个不同群体MHC ⅡB基因序列多态性和遗传分化

利用一对特异性引物MHC 2b-snp-sf和MHC 2b-snp-sr,从广东省3个不同罗非鱼养殖地区（以下分别称高要群体,茂名群体,惠州群体）采集的137尾尼罗罗非鱼（Oreochromis niloticus,GIFT strain）的个体基因组中扩增出长度为775-796 bp的片段。克隆后测序,序列分析结果显示：775-796 bp的核苷酸序列中共检测出62个简约信息位点,49个单碱基突变位点。112个多态位点中,增添/缺失位点34个,转换位点44个,颠换位点33个,另外一个位点既出现转换（C/T）又出现颠换（A/T）。137尾个体的751个阳性克隆的测序结果分析表明,751条序列分属于4个等位基因。其中,等位基因Orni-DAB＊0101在3个群体中所占比例最高（85.2%）。等位基因的分布及群体内遗传多样性参数分析表明：高要、茂名群体遗传多样性较丰富,惠州群体遗传多样性较低。群体间的分化指数（FST值）、平均基因流（Nm）、分子方差分析（AMOVA）和平均K2-P遗传距离均表明3个养殖群体间存在广泛的基因交流,未发生群体间的遗传分化。该研究结果提供了广东地区养殖尼罗罗非鱼MHCⅡB基因的部分遗传信息,为尼罗罗非鱼抗病品系的选育提供理论依据。     

适应玉米的溶磷细菌筛选及其对玉米生长的影响

从石灰性土壤中分离获得4株高效溶磷细菌X5、X6、Z4和Z8,研究其生物学特征,探索其单独及复合的溶磷促生潜能。研究发现菌株X5、X6、Z4和Z8均可以利用玉米根系分泌物作碳源生长。菌株X6和Z4均能产生吲哚乙酸(IAA)和铁载体,菌株Z8可产生IAA不产生铁载体,菌株X5可产生铁载体不产生IAA。盆栽试验结果表明,接种单一溶磷菌及4株菌复合处理均可促进玉米生长,但复合菌群的溶磷促生效果显著高于单一菌株。通过16S r RNA基因序列分析研究菌株的分类地位,初步鉴定X5、X6、Z4、Z8分别为荧光假单孢菌(Pseudomonas fluorescens)、草假单胞菌(Pseudomonas poae)、巨大芽孢杆菌(Bacillus megaterium)和枯草芽孢杆菌(Bacillus subtilis)。     

磷细菌筛选及其对土壤无机磷转化的影响

从潮土、水稻土、砂姜黑土和石灰土等土壤的植物根际土壤和根中分离了86株磷细菌,通过液体摇瓶培养3d,培养液水溶磷含量为4.2～387.3mg/L,水溶磷含量与培养液pH呈显著负相关(R2=0.621 6)。用筛选出的1株磷细菌(HCW115)进行玉米盆栽试验,结果表明,磷细菌处理的玉米干物重和吸磷量与对照相比分别增加了37.5%和40.2%,达到显著差异。磷细菌对土壤Al-P、Fe-P和O-P转化无明显影响,但可以促进土壤Ca2-P、Ca8-P和Ca10-P向有效磷转化而被玉米吸收,与原土相比,Ca2-P、Ca8-P和Ca10-P含量分别减少了74.9%,12.3%和1.51%。     

吸附法固定光合细菌技术产氢能力的研究

研究了吸附法固定光合细菌的产氢能力,结果表明采用不同的吸附材料固定光合细菌,其产氢能力与游离态细菌相比均有提高.产氢能力最好的是大粒经陶粒,平均总产氢量达到452.7 mL,是对照组游离态细菌总产氢量的2.16倍,平均产氢速率是对照组的1.95倍,小粒径陶粒的产氢能力次之,平均总产氢量是对照组的1.72倍,平均产氢速率是对照组的1.73倍,沙砾和大孔树脂的产氢能力低于陶粒,但优于游离态细菌,产氢量分别是对照组的1.43倍和1.09倍,产氢速率分别是对照组的1.34倍和1.08倍.研究还发现采用吸附法固定光合细菌进行产氢,不仅能提高产氢能力,而且能提高氢气含量,延长产氢时间.     

与黑鹰嘴豆(Vigna mungo L.)共生的VA菌根、解磷细菌及根瘤菌在田间条件下的协同作用

A field experiment was conducted at the G.B.Pant University Research Station,Ujhani(U.P.)in rainy (Kharif) season of the year 1994-1995 to study the effect of Rhizobium,VAM(vesicular arbuscular myc-orrhiza) and PSB(phophate solubilizing bacteria) inoculation,with and without P,on blackgram(Vigna mungo L.) seed yield.Phosphorus application in sol with medium P content(5.4 mg kg^-1) increased nodulation,grain yield,N and P in plant and grain over no phosphorus control.Forty kilograms of P2O5 each hactare recorded an increase of 20.6% in nodule dry weight,significant increases of 0.35 g kg^-1 in N concentration and 1.28 g kg^-1 in P concentration of plant over 20 kg P2O5 ha^-1 ,Similar significan increases of 0.5 g kg^-1 in grain yield and 0.54 and 0.23 g kg^-1 in N and P concentrations of the grain,respectively,over 20 kg P2O5 ha^-1 were also obtained with higher dose.Inoculation of Rhizobium VAM PSB at all the stages of plant growth recorded maximum increaes in all the parameers studied .Dual inoculation of Rhizobium with either VAM or PSB was generally significan in the effect and better than that of VAM PSB,however,P accumulation in plat and grain was more with VAM PSB.A mong single inoculations,Rhizobium gave highest and 21.0% more nodule number,34.7% more nodule dry mass,0.73 g kg^-1 more N in grain and 4.2% higher grain yield over PSB.PSB.however,registered significant increases in P concentration in plant and grain over VAM and Rhizobium.     

Phosphate-solubilization activity of bacterial strains in soil and their effect on soybean growth under greenhouse conditions

The efficiency of 13 phosphate-solubilizing bacteria (PSB; four Burkholderia sp., five Enterobacter sp., and four Bradyrhizobium sp.) was assessed in a soil plate assay by evaluating soil phosphorus (P) availability. A commercial argentine strain, Pseudomonas fluorescens, was used for comparing solubilizing activity. Burkholderia sp. PER2F, Enterobacter sp. PER3G, and Bradyrhizobium sp. PER2H strains solubilized the largest quantities of P in the soil plate assay after 60 days as compared with the other strains, including the commercial one. The effect of PSB inoculation on growth and nutrient uptake of soybean plants was also studied under greenhouse conditions. Plants inoculated with Burkholderia sp. PER2F had the highest aerial height and showed an appropriate N/P ratio. However, none of the PSB increased P uptake by plants. This suggests that PSB inoculation does not necessarily improve P nutrition in soybean, nor was there any relationship between P availability in the soil plate assay and P content in the soybean shoot in the greenhouse. We concluded that the selection of efficient PSB strains as possible inoculation tools for P-deficient soils should focus on the integral interpretation of soil assays, greenhouse experiments, and field trials.     

Oral administration of kefiran induces changes in the balance of immune cells in a murine model

The aim of the present study was to evaluate the effect of the oral administration of kefiran on the balance of immune cells in a murine model. Six week old BALB/c mice were treated with kefiran (300 mg/L) for 0, 2 and 7 days. Kefiran treatment increased the number of IgA+ cells in lamina propria after 2 and 7 days. Percentage of B220+/MHCII(high) cells in mesenteric lymph nodes (2 days) and Peyer's patches (7 days) was higher compared to untreated control mice. An increase of macrophages (F4/80+ cells) was observed in lamina propria and peritoneal cavity (2 and 7 days). In contrast, at day 7, macrophage population decreased in Peyer's patches. These results show the ability of kefiran to modify the balance of immune cells in intestinal mucosa. This property could be highly relevant for the comprehension of the probiotic effect attributed to kefir.     

Tricalcium phosphate is inappropriate as a universal selection factor for isolating and testing phosphate-solubilizing bacteria that enhance plant growth: a proposal for an alternative procedure

Literature analysis and chemical considerations of biological phosphate solubilization have shown that the commonly used selection factor for this trait, tricalcium phosphate (TCP), is relatively weak and unreliable as a universal selection factor for isolating and testing phosphate-solubilizing bacteria (PSB) for enhancing plant growth. Most publications describing isolation of PSB employed TCP. The use of TCP usually yields many (up to several thousands per study) isolates “supposedly” PSB. When these isolates are further tested for direct contribution of phosphorus to the plants, only a very few are true PSB. Other compounds are also tested, but on a very small scale. These phosphates (P), mainly Fe-P, Al-P, and several Ca-P, are even less soluble than TCP in water. Because soils greatly vary by pH and several chemical considerations, it appears that there is no metal-P compound that can serve as the universal selection factor for PSB. A practical approach is to use a combination of two or three metal-P compounds together or in tandem, according to the end use of these bacteria—Ca-P compounds (including rock phosphates) for alkaline soils, Fe-P and Al-P compounds for acidic soils, and phytates for soils rich in organic P. Isolates with abundant production of acids will be isolated. This approach will reduce the number of potential PSB from numerous isolates to just a few. Once a potential isolate is identified, it must be further tested for direct contribution to P plant nutrition and not necessarily to general growth promotion, as commonly done because promotion of growth, even by PSB, can be the outcome of other mechanisms. Isolates that do not comply with this general sequence of testing should not be declared as PSB.     

Comparative efficiency of phosphate-solubilizing bacteria under greenhouse conditions for promoting growth and aloin-A content of Aloe barbadensis

This study was conducted with Aloe barbadensis in order to investigate the efficacy of four phosphate-solubilizing bacteria (PSB), Pseudomonas synxantha 10223, Burkholderia gladioli 10242, Enterobacter hormaechei 10240 and Serratia marcescens 10241 to solubilize Mussorie rock phosphate (MRP) and to evaluate its effects on growth, soluble P content and P uptake compared with control, i.e. uninoculated plants. Pot experiments were conducted in a greenhouse, in soil supplemented with MRP. Each PSB treatment showed different effects on different plant growth parameters. The maximum increase in leaf length (23.7%), total number of leaves (33.33%) and dry rind weight (69.10%) was observed in plants treated with P. synxantha 10223 compared with control. Whereas, maximum increase in root length (23.43%), fresh leaves weight (79.03%), dry gel weight (113.08%) and total gel volume (112.10%), was observed in plants treated with S. marcescens 10241 compared with uninoculated plants. Maximum increase in aloin-A content [114.92% (per g dry gel weight) and 322.32% (per plant dry gel weight)] was observed in plants treated with P. synxantha 10223 compared with control plants. Root colonization by inoculated PSB as estimated by RAPD technique showed that all PSB were able to survive in the rhizosphere of Aloe plants.     

Differential interaction between two Glomus intraradices strains and a phosphate solubilizing bacterium in maize rhizosphere

Arbuscular mycorrhizal (AM) fungi and phosphate solubilizing bacteria (PSB) have a positive effect on plant productivity primarily through increasing phosphate availability. In order to study the interaction between AM fungi and PSB, we used Bacillus megaterium, a PSB isolated from the sterilized surface of AM germinated spores, and two strains of the AM fungus Glomus intraradices with different mycelial architecture. A greenhouse experiment was designed with maize as host plant with the addition of tribasic calcium phosphate. We tested the hypothesis that PSB, intimately linked with AM fungi, could interact differentially with the two AM strains. We concluded that inoculation with the PSB positively affected maize mycorrhization. Insoluble phosphate alone did not influence the AM extraradical mycelium (ERM) length and maize mycorrhization when bacteria were not inoculated. The results provide evidence that the adverse effect on infectivity for some AM strains might be caused by solubilized phosphorus release to the rhizosphere by PSB. Differences related to the mycelium architecture of each AM strain were observed: the density of PSB in rhizosphere soil was significantly higher only with the GA8 strain coinciding with the highest values of maize biomass. The density of bacteria associated with GA8 mycelium could be the result of the transfer of photosynthates through the rhizosphere; this close contact would favor the persistence of the intimate relationship between PSB and AM hyphae. In the bacteria-free treatments, soil adherence was not significantly altered. Although the highest development of ERM occurred with GA5, plants inoculated with GA8 showed the highest values for soil adherence. This may be due to the AM mycelium which modifies bacterial persistence in the rhizosphere and consequently soil adherence. Our results show that for potential applications, some characteristics of the AM strains are key in the selection of the AM fungi–PSB combinations. These include the tolerance to soluble phosphorus, the rate of root colonization, and ERM development that favors the persistence of bacteria in rhizosphere soil.     

Enhancement of maize plant growth with inoculation of phosphate-solubilizing bacteria and biochar amendment in soil

Maize plant has an absolute requirement of nutrients (N, P, and K) for growth and development. The microbial application can facilitate in addressing limited access to chemical fertilizer concern. Moreover, biochar and phosphorus-solubilizing bacterial (PSB) community can contribute together in nutrient availability. Both have the P-supply potential to the soil, but their interaction has been tested less under semiarid climatic conditions. The purpose of the study was to evaluate the potential of biochemically tested promising PSB strains and biochar for maize plant growth and nutritional status in plant and soil. Therefore, two isolated PSB strains from maize rhizosphere were biochemically tested in vitro and identified by 16S rDNA gene analysis. The experiment was conducted in the greenhouse where the plant growth and nutrient availability to the plants were observed. In this regard, all the treatments such as PSB strain-inoculated plants, biochar-treated plants, and a combination of PSBs + biochar-treated plants were destructively sampled on day 45 (D₄₅) and day 65 (D₆₅) of sowing with four replications at each time. PSB inoculation, biochar incorporation, and their combinations have positive effects on maize plant height and nutrient concentration on D₄₅ and D₆₅. In particular, plants treated with sawdust biochar + Lysinibacillus fusiformis strain 31MZR inoculation increased N (32.8%), P (72.5%), and K (42.1%) against control on D₆₅. Besides that, only L. fusiformis strain 31MZR inoculation enhanced N (23.1%) and P (61.5%) than control which shows the significant interaction of PSB and biochar in nutrient uptake. PSB and biochar have the potential to be used as a promising amendment in improving plant growth and nutrient absorption besides the conventional approaches.