无指盘臭蛙皮肤抗茵肽的分离纯化与氨基酸序列测定

在抗菌活性跟踪下对四川冕宁产无指盘臭蛙（Odorranagrahami）皮肤分泌物进行一系列层析分离，最终纯化得到4种抗菌肽，分别命名为Odorgrin A、Odorgrin B、OdorgrinC和OdorgrinD．质谱测得单一同位素分子量恤分别为3364．8、3817．9、4859．4和3287．9．Edman降解法测得OdorgrinA的氨基酸序列为GLLDTFKNLALNAAKSAGVSV—LNSLSCKLSKTC．将以上结果与国内外对该物种抗菌肽研究的已有结果进行了比较分析，证明本研究分离到的4种抗菌肽与前人分离到的部分抗菌肽间具有一致性．     

无指盘臭蛙皮肤抗菌肽Odorgrin A真核表达载体的构建与转化

根据无指盘臭蛙(Odorrana grahami)皮肤抗菌肽Odorgrin A的氨基酸序列,合成了以酵母偏爱密码子编码的Odorgrin A基因片段.目的片段从合成质粒上用XhoΙ和EcoRΙ双酶切下后,与经同样限制酶酶切的pPIC9K载体连接而成表达载体pPIC9K-Odo A.PCR扩增、酶切及测序检测的结果表明表达载体构建成功.线性化的pPIC9K-OdoA经电击法转化毕赤酵母(Pichia pastoris)GS115宿主菌,营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测的结果表明,表达载体pPIC9K-Odo A成功地转化并整合入酵母基因组.用甲醇对具遗传霉素G418高抗性的Odorgrin A重组酵母菌进行诱导表达,取酵母发酵液上清经SDS-PAGE电泳检测,结果初步表明,整合进酵母基因组的抗菌肽Odorgrin A基因已获得分泌表达,表达产物相对分子质量为3×103～4×103,与理论值相近.图10参17     

小皱蝽抗菌物质的诱导及抗菌肽AMP-2的分离纯化和特性

针刺小皱蝽成虫腹部,诱导免疫防御反应,小皱蝽成虫组织经过浸提、浸提液TFA酸化、超速离心、超滤、固相萃取等分离步骤,平板生长抑制试验检测抗菌活性.在C18Sep-Pak固相萃取柱上,40?N洗脱组分为初样品,经RP-HPLC、GPC-HPLC及填料孔径更小的反相色谱柱纯化,得到0.452mgAMP2样品.琼脂孔扩散法检测AMP2抗菌活性,回收率为4.04%.抗菌肽AMP2在SuperdexPeptide凝胶过滤柱上测得分子量(Mr)为3.2×103,茚三酮反应紫色.AMP2具有一定热稳定性,在pH3～8的乙酸铵缓冲液中抗菌活性强,对检测的动植物病原菌具有广谱抗菌活性,对金黄色葡萄球菌、大肠杆菌D22、枯草芽孢杆菌的MIC值为2.5μg/mL,对四联球菌的MIC值为5.0μg/mL,AMP2可能为富含脯氨酸的小分子抗菌肽.图3表2参11     

农药在土壤中的吸附

对农药涕灭威,林丹和氟乐灵在三种不同类型土壤,即红泥沟土、沙河士和百花山土中的吸附行为进行了研究.分别用平衡法和柱法测得了它们的吸附常致,给出了吸附等温线,并对测得结果进行了讨论.     

利用果胶培养基分离番茄根际细菌

采用平板稀释和PCR-DGGE相结合的方法,比较了4种培养基(果胶富营养培养基PM,只含果胶一种营养的培养基PA,添加果胶的寡营养培养基YPP以及与YPP营养成分一致但不添加果胶的寡营养培养基YPG)分离番茄根际细菌的能力.结果显示:PA培养基能够分离到42种形态的细菌;YPP培养基可以分离到最高的细菌菌落数,分离获得的种类比PA少;YPP培养基分离获得细菌的种类和菌落数量比YPG培养基要多.聚类分析也显示只有果胶一种营养的PA培养基能分离到最多种类,分离获得的菌群与自然环境中微生物群落最相似.研究结果表明添加番茄根际主要分泌物——果胶到培养基中可以提高培养基分离细菌的能力.     

茄子植物内生细菌群落结构与多样性

采用平板分离纯化不同来源地茄子健株和青枯病株内生细菌,利用细菌脂肪酸鉴定技术进行鉴定.共获得内生细菌18属28种,健株共分离到内生细菌15属23种,含量为5.96×105CFU·g-1.病株分离到内生细菌14属16种,含量为3.191×108CFU·g-,其中青枯雷尔氏菌含量为3.187×108CFU·g-.根部分离到的内生细菌最多,其次为茎部,叶部最少.不同种植地来源的茄子健株内生细菌的种类数量有较大差异,来自晋江的健株分离到13属17种,来自东洋的健株分离到7属9种,而来自福清的健株仅分离到3属4种.来自晋江的植株内生细菌的SIMPSON(D)优势度指数和SHANNON(H1)多样性指数分别为0.963 2和3.572 5,均高于来自东洋植株(D:0.9444,H1:2.725 5)和福清植株(D:0.833 3,H1:1.5).而均匀度则以来自东洋植株最高(0.970 8),来自晋江和福清的植株分别为0.9654和0.9464.不同生长环境下不同生长状态的茄子植株内生细菌群落结构与多样性存在较大的差异.该研究对于通过微生物标记植物生长的生态环境具有重要的实验意义,对于科学说明生防机理具有重要作用.     

不同培养条件对罗非鱼肠道乳酸菌分离的影响

为了解罗非鱼肠道乳酸菌群多样性和优势菌种,促进乳酸菌在罗非鱼养殖中的应用,采用MRS、M17、TPY、Elliker四种培养基,分别在有氧、厌氧以及30℃、37℃和45℃温度条件下分离罗非鱼肠道乳酸菌,分析不同培养基、培养温度、有氧和厌氧条件对罗非鱼肠道乳酸菌分离的影响;并对分离到的菌株进行分子鉴定和16S r RNA序列分析,构建系统发育树,比较不同分离条件下分离肠道乳酸菌的遗传多样性.结果表明,不同培养基对罗非鱼肠道乳酸菌的分离效果有一定的差异,其中MRS培养基的分离效果最好,且遗传多样性最高;37℃较30℃对乳酸菌的分离更加有利,45℃未分离到乳酸菌;有氧条件下共分离出8种33株乳酸菌,主要为杆菌;厌氧条件下得到10种24株乳酸菌,主要为球菌.共分离到植物乳杆菌(Lactobacillus plantarum)29株,可能是罗非鱼肠道优势乳酸菌种.本研究可为开发水产养殖肠道乳酸菌提供依据,并推广到其他种类的鱼肠道乳酸菌的分离和鉴定.     

水稻内生粘着剑菌的分子系统发育分析

通过16S rRNA基因和看家基因recA、atpD、rpoB的序列测定与分析,及(G+C)mol%测定等技术,对分离自河北省滦南县水稻根内的两株细菌进行分子系统发育学研究.根据系统进化树构建及其同源性分析,可确定所分离的两株水稻内生细菌属于剑菌属(Ensifer)系统发育分支;供试菌株的各保守序列与粘着剑菌(E.adhaerens)模式菌种同源性最高,除recA为97.2%外,其他均为99%～100%,可以确定供试菌属于E.adhaerens;对所分离的菌株与大豆、菜豆、三叶草、苜蓿等4种豆科植物进行结瘤试验,结果显示其中一株J3-N55可与苜蓿结瘤.野生型的E.adhaerens菌株与豆科植物结瘤的报道尚为鲜见;本文首次报道从水稻植株内分离到E.adhaerens菌株.     

质子转移反应飞行时间质谱仪(PTR-ToF-MS)高时间分辨在线测定广州环境空气还原性硫化合物

采用高分辨质子转移反应飞行时间质谱仪(PTR-ToF-MS)对环境空气中还原性含硫化合物(RSCs)进行高时间分辨率在线测量.时间分辨率为2 s时,硫化氢(H_2S)、甲硫醇(MeSH)、二甲硫(DMS)和二甲二硫(DMDS)的检测限分别为25、26、22、80 ng·m~(-3);时间分辨率为10 min时,4种RSCs的检测限全面优于离线测量方法.标准样品10次重复测量表明,RSCs相对标准偏差3%,与真实浓度相对偏差在±5%以内.通过理论计算得到的绝对定量结果与外标法测得结果的比较,对绝对定量时理论计算的RSCs与H_3O~+反应常数k值进行了校正.利用校正后k值对广州环境空气RSCs进行绝对定量,结果与外标法测量结果相对偏差4%; 2017年2月对广州环境空气RSCs进行了为期一周的初步在线观测,测得H2S、DMDS、DMS和MeSH的浓度水平分别为501±155、1026±388、215±35、120±25 ng·m~(-3).RSCs日变化呈现差异,H2S、MeSH和DMS浓度日变化规律不明显,而其DMDS则呈现出较为明显的日变化特征.     

基于不同评估方法的养殖池塘富营养化环境成本研究——以上海青浦地区常规鱼类养殖池塘为例

采用物料平衡法、置换成本法、条件价值评估法（CVM）和COD价格法对池塘养殖环境成本进行评估。结果显示,基于4种评估方法估算得到的池塘养殖环境成本由大到小依次为置换成本法（4 400元.a-1.hm-2）、COD价格法（4 191.99元.a-1.hm-2）、物料平衡法（3 925.76元.a-1.hm-2）和CVM法（2 007.8元.a-1.hm-2）,认为在不考虑时间和经济成本的前提下,基于COD价格法测算得到的结果可能最接近于环境成本的真实值。采用4种评估方法测得的环境成本占常规鱼类养殖经济总收益的6%～14%,表明采取相关措施防治水产养殖环境污染很有必要。     

High amounts of free aromatic amino acids in the protein-like fluorescence of water-dissolved organic matter

Fluorescence spectroscopy is widely used to study water pollution. The fluorescence of water natural organic matter can be classified into two groups: the protein-like fluorescence originating from aromatic amino acids and the humic fluorescence originating from humic substances. Actually, the precise molecular origin of the protein-like fluorescence is unknown because this fluorescence may be caused by either free amino acids, peptides or proteins. Therefore, we studied the molecular origin of the protein-like fluorescence of Suwannee River natural organic matter and fractions A, B and C + D obtained by size exclusion chromatography/polyacrylamide gel electrophoresis. Fractions were analyzed by reversed-phase high-performance liquid chromatography. The electrophoretic mobilities of fractions varied in the order C + D > B > A and the molecular size in the opposite order. Our results show that the protein-like fluorescence is almost exclusively located in high molecular size fraction A and medium molecular size fraction B. Retention times and fluorescence emission spectra of authentic free aromatic amino acids tyrosine and tryptophan were identical with the retention times and emission spectra of several chromatographic peaks of fractions A and B. More than 50 % of the protein-like fluorescence is due to free aromatic amino acids incorporated in water natural organic matter.     

桦木内生真菌的分离与代谢产物的抑菌活性

通过琼脂块法和滤纸片法,对白桦Betula platyphylla、棘皮桦B.dahurica、硕桦B.costata和柴桦B.fruticosa）的内生真菌产生抑菌物质的特性进行研究,结果表明分离自白桦3年生枝条的内生真菌拟茎点霉Phomopsis sp.BP103381表现出较强的抑菌物质形成能力。采用正交设计优化了BP103381的培养条件,在含有40 g/L蔗糖、3 g/L NaNO3、1 g/LMgSO4的改良查氏液体培养基中,在pH值6.5、28℃条件下发酵培养9 d,有利于抑菌活性物质的形成。BP103381产生的抑菌活性物质具有较好的温度稳定性,但是酸碱变化对抑菌物质的活性影响较大,BP103381产生的抑菌物质在pH为5.0~7.0时活性较强。     

紫羊茅根中铜结合肽的分离和纯化

利用１次ＳｅｐｈａｄｅｘＧ５０凝胶过滤和２次ＱＡＥＳｅｐｈａｄｅｘＡ２５离子交换层析的方法,从最后６ｄ用ＣｕＣｌ２处理（ρ＝２０ｍｇ／Ｌ）、２０ｄ龄的紫羊茅（Ｆｅｓｔｕｃａｒｕｂｒａｃｖ．Ｍｅｒｌｉｎ）根中分离纯化了１个铜结合肽（ＣｕＢＰ２ａ）,该肽在ＳｕｐｅｒｄｅｘＰｅｐｔｉｄｅＨＲ１０／３０预装柱的ＦＰＬＣ凝胶过滤中在λ２５４ｎｍ处只出现一个峰,经分子量标准曲线估计,其Ｍｒ约为１４００,在ＤｅｌｔａＰａｋＣ１８的反相ＨＰＬＣ中,在λ２５４ｎｍ处也只有１个紫外吸收峰值,这都说明该肽已经达到了较高的的纯度．通过光谱分析,发现该肽的最大紫外吸收值在２４８．１ｎｍ处．对Ｃｕ结合物在植物体内的存在状态进行了讨论．     

Hematoxicity and hepatoxicity attributed to zinc and cadmium in the Norwegian rat

This study was carried out to determine changes in the blood and liver of rats given oral doses of cadmium (Cd) and zinc (Zn). Thirty 3 month old rats were kept six to a compartment in a well-demarcated five compartment cage labeled A, B, C, D, and E. After 2 weeks of acclimatization, and with ad libitum administration of water and feed, group A and B rats were, respectively, exposed to 100 mg CdSO₄ and 2200 mg ZnSO₄, respectively, mixed with their feed. Group C rats were fed with a combination of 100 mg CdSO₄ and 2200 mg ZnSO₄ mixed with their feed, while group D rats were exposed to 100 mg CdSO₄ mixed with their feed for 48 h, and after they were given 2200 mg ZnSO₄ for 96 h. Group E rats served as control and were fed a normal diet. Analysis of blood samples after 5 days showed that in rats of groups A, B, C, and D, WBC count increased from 6800 to 12,400 mm^?3 and platelets from 26,000 to 88,000 mm^?3. Reduction in blood counts were found for RBC from 13.8 to 4.7 × 10⁶ mm^?3, hemoglobin from 15.6 to 11.4 dL^?1, and PCV from 47 to 34%. ESR was the same 1.0 mm h^?1 in all groups. Severe damage to the liver was also shown by severe degeneration of hepatocytes and increase in Kuppfer cells. The above observations were mildest in the liver of group C rats. A reduction of body weight was observed in all treatment groups. Cd and Zn were found to be hematoxic and hepatotoxic in the Norwegian rat with Cd exhibiting greater toxicity than Zn. Toxicity of Cd was suppressed by Zn only when both metals were admistered at the same time, but a delay in intake of Zn (after 48 h) had little effect on Cd toxicity.     

杠板归的化学成分

采用柱层析方法从蓼科药用植物杠板归(Polygonum Perfoliatum)分离了21个化合物(1～21),通过MS与NMR数据鉴定这些化合物为α-tocopherolquinone (1), 7'-dihydroxymatairesinol (2), (24S)-24-ethylcholesta-3β,5α,6α-triol (3), 4-dihydroxy-5,7-dihydroxy-4-(4-hydroxyphenyl) coumarin (4), quercetin (5), cucurbitacin Ⅱa (6), cucurbitacin U (7), iotroridoside A (8), pokeweedcerebroside 5 (9), bonaroside (10), helonioside A (11), helonioside B (12), lapathoside D (13), vanicoside B (14), vanicoside C (15), vanicoside F (16), asteryunnanoside F (17), saikosaponin M (18), hydropiperoside (19), quercetin-3-O-β-D-glucuronide-6"-butyl ester (20), quercetin-3-O-β-D-glucuronide-6"-methyl ester (21). 化合物1～19(除4,5,12)为首次从该植物中分离得到,化合物1～11(除4,5)和17～19为首次从该属植物中分离得到.图1参22     

Evaluation of drought tolerance of new grapevine rootstock hybrids

The drought tolerance is a very important property of grapevine rootstocks. For that reason the breeding and selection of new rootstock varieties is focused also on the evaluation of their drought tolerance. In this experiment, altogether 20 new hybrids and 4 existing rootstock varieties were compared and evaluated. The experimental scheme involved 3 variants of water supply. Evaluated were the following properties: growth intensity of annual shoots, CCI (chlorophyll content index) and visual characteristics of plants. The most resistant were hybrids from the pedigree groups C (Binova x B?rner), D /Binova x/(Binova x Teleki 5C/) x B?rner/, and F (Teleki5 Cx B?rner). The following hybrids were classified as drought-tolerant: 17-1-6 (C); 17-1-9 (C); 17-6-2 (C); 17-6-9 (C); 17-8-2 (D) and 9-20-1 (F). Based on obtained experimental results and also on correlations existing between individual traits it can be concluded that practically all traits under study may be used when evaluating the resistance of plants to drought. The obtained results indicated that the B?rner rootstock (and thus also the species Vitis cinerea) can be used as a suitable genetic resource for the purpose of the breeding grapevine rootstocks for tolerance to drought.     

对甘薯茎线虫具有杀虫活性的苏云金芽胞杆菌筛选及特性鉴定

为筛选对甘薯茎线虫具有高毒力的苏云金芽胞杆菌(Bacillus thuringiensis,Bt)菌株,提取12株供试Bt菌株的晶体蛋白,溶解后加入到甘薯茎线虫悬浮液中,统计3 d和7 d后甘薯茎线虫的死亡率.经初筛和复筛后得到一株对甘薯茎线虫具有高毒力的Bt菌株YBT-008,其对甘薯茎线虫的LC50值为203.76μg/mL.研究了YBT-008的生物学特性,结果表明,YBT-008在LB培养基上培养约12 h后进入稳定期,并且伴随芽胞的形成产生卵圆形的伴胞晶体;SDS-PAGE分析表明YBT-008可产生多种待鉴定的晶体蛋白类型,质粒检测显示该菌株有5条质粒条带.YBT-008的获得为利用Bt防治甘薯茎线虫提供了新型菌株和基因资源.     

D.radiodurans kat A和kat B基因的结构分析与功能鉴定

以简并引物PCR获得的Cat酶基因片段探针,结合生物信息学方法,探明了耐辐射奇球菌（D.radiodu-rans)基因组中存在两个编码过氧化氢酶（Catalase,Cat)的基因：katA和B.katA和katB基因长2277bp和1611bp,各编码758和536个氨基酸,用pKK223-3表达载体连接katB基因,转入Cat酶缺陷型大肠杆菌（E. coliUM2)进行表达.katB基因表达产物具有Cat酶活性,电泳迁移位置与D.radiodurans CatB酶位置相符,并可重建E.coliUM2对低浓度H2O2损伤的耐受力。