基于DNA探针和FISH技术对两种有害浮游植物的检测研究

针对两种典型的有害浮游植物微小原甲藻和Takayama pulchellum(T.pulchellum)各设计了4条特异性探针,并采用基于PCR管载体的荧光原位杂交检测方法检测了这些探针的标记效果。实验结果表明,针对微小原甲藻和T.pulchellum核糖体大、小亚基DNA(LSU和SSU rDNA)的序列信息所设计的8条荧光标记探针均能够特异性地识别这些目标藻。各探针的标记效果有一定差异,经过标记的目标藻细胞在单种和自然水样混合样品中均可以通过荧光显微镜进行识别和区分。针对微小原甲藻和T.pulchellum的荧光原位杂交检测方法的建立将有助于对样品中这些目标藻进行快速准确地检测和监测。     

南麂列岛海域硅藻和甲藻群落的分布特征

于2006年4月~2007年3月采集浙江南麂列岛海域水样,研究了硅藻和甲藻群落在该海域的种类组成、生态分布特征和季节动态.共鉴定浮游植物93种,其中硅藻35属68种,甲藻11属21种.南麂海域浮游植物细胞丰度较高,年平均细胞丰度达到3.19105cellsL-1.硅藻集群的高峰区主要集中在夏季和秋季,细胞在表层水体的含量高于底层水体,调查期间共记录硅藻赤潮事件1起,发生于2006年8月,优势种类是中肋骨条藻.甲藻赤潮仅在春季（5月）发生,优势种类为原甲藻种类,包括三角棘原甲藻和东海原甲藻,分别在5月中旬和5月下旬先后引发赤潮,甲藻在表层水体含量高于底层水体.     

2010年夏季广西北部湾沿岸浮游植物分布特征及其与环境因子的相关性

2010年6月4日至26日在广西北部湾沿岸共设46个站位凋查分析浮游植物群落结构特征,以及浮游植物数量与环境因子的相关性。结果共鉴定出浮游植物70属149种(含变种和变型),其中硅藻41属106种,占总物种数的71.14%,占总细胞丰度的95.81%;甲藻18属32种,占总物种数的21.48%,占总细胞丰度的1.44%。浮游植物平均密度为61.10×104 cell/L,硅藻平均细胞丰度为58.54×104 cell/L,甲藻类平均细胞丰度为0.88×104cell/L。浮游植物密集区在防城港海区,此外在北海营盘附近海域有一个较高的密度区。浮游植物丰度在茅尾海和钦州港近岸较低,其余海区的浮游植物数量分布态势为近岸高、外海低。浮游植物主要优势种为尖刺拟菱形藻(Pseudo-nitzschia pungens)、中肋骨条藻(Skeletonema costatum)、拟旋链角毛藻(Chaetocerospseudocurvisetus)、菱形海线藻(Thalassionema nitzschioides)、丹麦细柱藻(Leptocylindrus danicus)。尖刺拟菱形藻、拟旋链角毛藻密集区主要在防城港海区,而中肋骨条藻密集区主要在北海海区。浮游植物数量与温度、盐度及硝酸盐、亚硝酸盐、氨盐和硅酸盐无相关性,北海海区和防城港海区浮游植物丰度与活性磷酸盐呈显著正相关,磷在以上两个海区成为浮游植物生长的限制因子。     

海洋甲藻包囊及沿革

某些甲藻在面对不良环境时,机体会采取一定的措施——失去鞭毛、积累营养物质形成甲藻包囊沉入水层底部,一旦周围环境适宜,沉积物中甲藻包囊便可以萌发形成营养细胞再次进入水体.甲藻包囊的大量萌发极易形成甲藻优势种爆发,进而出现有害藻华现象.作为甲藻生活史中的一个重要阶段,包囊对甲藻种群的生存与发展具有重要的作用.甲藻包囊组成与分布可以间接反映一段时期内上层水体的营养状况,继而推测出调查海域是否有藻华爆发的潜在威胁.本文主要阐述了甲藻包囊的基本形态分类和术语,甲藻包囊沿革及其国内外研究现状,并对甲藻包囊与上层水体浮游植物的相互关系进行讨论.     

常见赤潮藻类的变性梯度凝胶电泳分析

为了解我国典型有毒有害赤潮藻类的变性梯度凝胶电泳( DGGE)指纹图谱以及DGGE技术在赤潮藻类分析鉴定中的作用,本课题组运用DGGE技术,研究了我国沿海7种重要赤潮藻类的单一种类以及混合种类样品的18S rDNA V3区的DGGE指纹图谱,并且对2009年10月底在广东省珠海海域发生的双胞旋沟藻(Cochlodinium gemtnatum)赤潮样品进行了DGGE分析.结果表明,DGGE技术能够对环节环沟藻、海洋原甲藻、血红哈卡藻、锥状斯氏藻、中肋骨条藻、塔玛亚历山大藻、双胞旋沟藻7种常见的海洋赤潮藻类具有较好的区分效果,同时监测出赤潮样品中双胞旋沟藻、血红哈卡藻、环节环沟藻和海洋原甲藻4种优势种,种类组成与显微镜观察结果具有较好的一致性.结果说明DGGE技术可作为一种辅助方法对赤潮藻类进行分类鉴定.     

泉州湾虾池浮游植物种类多样性研究

2003年11月～2004年5月对福建泉州湾东海虾池进行定点采样,并分析其浮游植物的种类多样性和种群动态.结果表明,泉州湾虾池共有浮游植物42属166种,硅藻为优势种群,共35属154种;甲藻3属6种.新月筒柱藻Cylindrotheca closterium、微小小环藻Cyclotella caspia、狭窄双眉藻Amphora angusta、菱形藻属Nitzschia为主要优势种,它们在浮游植物群落总细胞数中的百分比平均高达34.8%.通过对水温、营养盐、浮游植物种群密度及群落的多样性指数和均匀度的分析,发现虾池高强度的排污排废所引起的富营养化已很大程度影响了浮游植物的群落结构和时空分布,使生物多样性和均匀度下降,少数优势种类大量繁殖.硅藻和甲藻赤潮的发生机会增多.     

泉州湾虾池浮游植物种类多样性研究

2003年11月～2004年5月对福建泉州湾东海虾池进行定点采样，并分析其浮游植物的种类多样性和种群动态．结果表明，泉州湾虾池共有浮游植物42属166种，硅藻为优势种群，共35属154种；甲藻3属6种．新月筒柱藻Cylindrotheca closterium、微小小环藻Cyclotella caspia、狭窄双眉藻Amphora angusta、菱形藻属Nitzschia为主要优势种，它们在浮游植物群落总细胞数中的百分比平均高达34．8％．通过对水温、营养盐、浮游植物种群密度及群落的多样性指数和均匀度的分析，发现虾池高强度的排污排废所引起的富营养化已很大程度影响了浮游植物的群落结构和时空分布，使生物多样性和均匀度下降，少数优势种类大量繁殖．硅藻和甲藻赤潮的发生机会增多．     

秦皇岛贝类养殖区4种贝类对浮游植物的摄食选择性

为了研究秦皇岛贝类养殖区滤食性贝类饵料来源和摄食选择性特点,于2019年5月对该海域的海水和4种贝类(紫贻贝、海湾扇贝、长牡蛎、栉孔扇贝)进行采样调查。利用显微镜检法和光合色素液相色谱法(HPLC)对海水和贝类胃容物中的浮游植物种群结构进行比较研究。结果显示,水体中浮游植物主要由硅藻、甲藻、隐藻组成,硅藻在丰度上占有绝对优势。光合色素HPLC检出水体中特征色素主要为硅藻、青绿藻和甲藻,而贝类胃容物中主要为甲藻和硅藻,少量青绿藻检出。四种贝类摄食选择性相似且不受水体中优势藻种的影响,主要以甲藻、硅藻中不占优势的藻种为食,对部分占优势藻种表现出避食性(中肋骨条藻和角毛藻),微微型浮游植物(青绿藻)对4种贝类的饵料贡献率较低。     

深圳福田红树林保护区浮游植物群落的季节变化及其生态学研究

对深圳福田红树林保护区浮游植物群落结构和季节变化进行研究.共鉴定到浮游植物5门25属75种,其中硅藻门21属68种,蓝藻门1属1种,甲藻门1属1种,绿藻门1属3种,裸藻门1属3种.密度的季节变化范围为1.0×106～5.0×106个/L,平均密度为2.7×106个/L.赤潮藻和耐污染特征的种类如威氏海链藻和微小小环藻等是浮游植物的主要成分.底栖性、附着性和淡水性的种类在浮游植物中经常出现.与以前的研究比较,福田红树林区水体浮游植物群落朝着种类个体变小、种类数减少、密度增加、耐污染种类增加的方向变化,这反映了该红树林区水体富营养化程度高,水质持续恶化.     

用PCR扩增裸甲藻和微小原甲藻rRNA基因的方法研究

以赤潮种裸甲藻(Gymnodinium sp．)和微小原甲藻(Prorocentrum minimum)为试验材料，以不同方法提取其基因组并进行纯化，然后采用PCR方法扩增其rRNA基因，包括18S，28S和ITS片断，并进行扩增条件的比较和优化，得到两种藻的最佳DNA提取条件和PCR扩增条件．裸甲藻和微小原甲藻的DNA提取宜采用改良的CTAB方法；并需对粗提取的DNA用CTAB方法进行纯化．两种藻的最适模板浓度为纯化后模板1．0～2．0μL；最适Mg^2 浓度为2．0μL(25mmol／L)；ITS引物PCB扩增的退火温度为50℃，而18S三对引物的退火温度均为55℃，28S的退火温度为54℃最为适宜。     

Identification of a novel DNA molecule associated with Tobacco leaf curl virus

A circular DNA molecule, designated as DNAβ, was identified in tobacco plants infected with Tobacco leaf curl virus (TLCV) isolates Y5 and Y8 by PCR using primers based on the conserved region of the two reported DNAβ sequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβ of Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβ comprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAβ consists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβ has little sequence homology to DNA-A of TLCV., except that it shares conserved TAATATTAC loop sequence with TLCV DNA-A. Sequence comparison showed that Y5 DNAβ shared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβ and Y8 DNAβ had relatively low sequence identity (51%–65%) with the reported DNAβ molecules associated with Ageratum yellow vein virus and Cotton leaf curl virus. The immunotrapping PCR and whitefly transmission tests showed that DNAβ molecule could be encapsidated in virus particle and transmitted by Bemisia tabaci. This is the first report of DNAβ associated with WTGs in China.     

QTL analysis of the rice seedling cold tolerance in a double haploid population derived from anther culture of a hybrid betweenindica andjaponica rice

A doubled haploid population, derived from anther culture of F₁ hybrid between a typicalindica cv. and ajaponica cv. has been used to investigate the seedling cold tolerance (SCT) in growth cabinet. By dynamically analyzing every day’s survival percentages of the parents and DH lines under 7-d cold plus 9-d normal temperature condition, the quantitative trait loci (QTLs) for SCT have been mapped based on a molecular linkage map constructed from this population. The results show that two parents had significant differences in SCT and the segregation of SCT in DH lines was basically a continuous distribution with most serious injury on the 6th d of the cold treatment. A total of 4 QTLs for SCT have been identified on chromosomes 1, 2, 3 and 4 respectively. The additive effects of qSCT-1, qSCT-2 and qSCT-3 have been contributed by thejaponica cv JX17, but that of qSCT-4 has been contributed by theindica cv ZYQ8. The mechanism of SCT seems complicated since the above 4 QTLs detected at different stages during the treatment. Further study on the genotypes for these SCT QTLs in the DH lines shows transgressive segregation. It is demonstrated that the lines with stronger SCT over JXI7 have 3–4 loci for SCT. Integration of these QTLs into an appropriate variety may lead to a successful rice breeding program for cold tolerance.     

Cloning and characterization of a gene encoding phagerelated tail protein (PrTP) of endosymbiont Wolbachia

Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.     

Tobacco curly shoot virus isolated in Yunnan is a distinct species of Begomovirus

Virus isolate Y1 was obtained from tobacco showing curly shoot symptoms in Baoshan, Yunnan Province. Whitefly transmission test and virion morphology observation showed that it is a begomovirus. In reactions with 14 monoclonal antibodies raised against begomoviruses, Y1 was readily differentiated from begomoviruses reported in China, Pakistan and India. The complete nucleotide sequence of DNA-A was determined, it contains 2746 nucleotides, with two ORFs in virion-sense DNA and four ORFs in complementary-sense DNA. Comparisons with total DNA-A, intergenic region and deduced amino acid sequences of individual ORFs showed that Yl is a distinct Begomovirus species, for which the name Tobacco curly shoot virus (TCSV) is proposed. The total DNA-A of TCSV is most closely related to that of Tomato leaf curl virus from India (85% sequence identity). In contrast, the deduced coat protein of TCSV is most like that of Cotton leaf curl virus 72b isolate from Pakistan (98% amino acid sequence identity).     

混合菌种发酵生产酱油的工艺研究

利用米曲霉(Aspergillus oryzae)和黑曲霉(Aspergillus niger)混合接种,并在制曲后期混合添加嗜盐片球菌(Pediococus halophilus)和高渗酵母进行多菌种发酵生产酱油实验研究。结果表明,混合发酵的菌种最佳培养时间为36～40h,最佳发酵温度为45～48℃。米曲霉和黑曲霉的接种比为90%∶10%时,全氮利用率提高13.2%,氨基酸生成率提高6.8%。将成熟曲拌以10%的食盐水,在pH值为6.5的环境下,混合添加嗜盐片球菌和高渗酵母参与发酵可以提高酱油产品中的醇类含量和酸类含量,改进酱油的风味。     

鸡蛋花鞘锈菌重寄生真菌的种类鉴定及其寄主范围测定

鉴定鸡蛋花鞘锈菌Coleosporium plumeriae重寄生真菌的种类,明确其寄主范围,为鸡蛋花锈病及其他植物锈病的生物防治提供依据。通过分离和纯化鸡蛋花锈病叶背夏孢子堆上的重寄生真菌,经单孢纯化获得12个单孢菌株,其菌落及分生孢子特征一致,菌落白色,分生孢子近圆形或卵圆形,单胞,无色,呈头状聚生在分生孢子梗顶端。选取菌株PR-CPH1作为代表,分析3个片段的核苷酸序列,PR-CPH1的ITS（MW505984）、SSU（MW505988）和LSU（MW505987）序列与Simplicillium subtropicum的ITS（AB603990）、SSU（LC496895）和LSU（LC496880）序列的一致性分别为99.00%、98.11%和98.11%。系统发育树分析发现,菌株PR-CPH1的ITS-SSU-LSU联合序列与S.subtropicum(菌株号:JCM18180)聚在同一个分支。结合形态特征和ITS、SSU、LSU的序列分析,鉴定鸡蛋花鞘锈菌重寄生真菌为S.subtropicum。测定S.subtropicum寄主范围的结果显示,在8种植物锈菌中,菌株PR-CPH1可重寄生鸡矢藤鞘锈菌C.paederiae、香茶菜鞘锈菌C.plectranthi和落叶松-杨栅锈菌Melampsora larici-populina。S.subtropicum为4种锈菌的重寄生真菌为首次报道。     

Cloning and sequence analysis of Sox genes in a tetraploid cyprinid fish, Tor douronensis

A PCR survey for Sox genes in a young tetraploid fish Tor douronensis （Teleostei： Cyprinidae） was performed to access the evolutionary fates of important functional genes after genome duplication caused by polyploldizatlon event. Totally 13 Soxgenes were obtained in Tordouronensis, which represent SoxB, SoxC and SoxE groups. PhylogeneUc analysis of Sox genes in Tor douronensis provided evidence for fish-specific genome duplication, and suggested that Sox19 might be a teleost specific Sox gene member. Sequence analysis revealed most of the nucleoUde substitutions between duplicated copies of Soxgenes caused by tetreploldlzatlon event or their orthologues in other species are silent substitutions. It would appear that the sequences are under purifying selective pressure, strongly suggesting that they represent functional genes and supporting selection against all null allele at either of two duplicated loci of Sox4a, Sox9a and Sox9b. Surprising variations of the intron length and similarities of two duplicated copies of Soxga and Sox9b, suggest that Tor douronensis might be an allotetreploidy.     

烟碱降解菌DAB2菌株的筛选、鉴定和降解特性

以烟碱为唯一碳源,从湖北省襄阳烟叶种植土壤中分离得到了一株烟碱降解菌,命名为DAB2,经形态观察、生理生化分析和16S rDNA序列分析,初步鉴定该菌株为中间苍白杆菌(O.intermedium).当培养基中烟碱质量浓度为1.0 g/L,DAB2菌株生长良好,烟碱降解率为88.42%;当烟碱质量浓度为3.0 g/L,降解率为52.46%;当烟碱质量浓度高于6.0 g/L时,菌株生长和烟碱降解率均呈下降趋势,烟碱降解率低于10.36%.DAB2菌株在烟碱质量浓度为1.0 g/L的烟碱培养基中培养,不同时间样品紫外扫描显示:0 h只有259nm处有吸收波峰,8 h时259nm处波峰降低,在230nm和290nm处出现了新峰,表明产生了中间产物.     

Identification of a disulfide bonded protein from cytoplasm ofBacillus subtilis

Conclusion In this study, P23 was found to be a disulfide-bonded cytoplasmic protein, abundant in late exponential phase and stationary phase cells, and was hardly detected in early exponential phase cells, the cells of sporulation process and spores. Therefore, synthesis of P23 was regulated by some specific factors or/and cellular environment. Conclusively, cytoplasm has a mechanism to catalyze the formation of disulfide bonds, which is consistent with Dermanet al. ’s conclusion from mutation experiment^[1].