大白菜、青花菜和叶用芥菜体细胞杂交种形态学与细胞学特性鉴定

 为拓宽十字花科蔬菜育种种质资源,以大白菜（Brassica campestris L. ssp. pekinensis）、青花菜（B. oleracea L. var. varitalica）和叶用芥菜（B. juncea L. Czern）的子叶、下胚轴为材料,分离、纯化原生质体,采用40% 聚乙二醇（Polyethylene glycol,PEG）融合法进行原生质体融合。融合细胞在附加0.2 mg · L^-1 2,4-D + 0.5 mg · L^-1 6-BA + 0.1 mg · L^-1 NAA + 0.1 mg · L^-1 激动素（kinetin）的改良K8p培养基中液体培养,当细胞分裂至8 ~ 10细胞期时,将分裂的细胞包埋于0.15%琼脂糖,然后在添加0.3 mol · L^-1蔗糖和2 mg · L^-1 6-BA + 2 mg · L^-1 玉米素（zeatin）+ 1 mg · L^-1 NAA + 0.5 mg · L^-1 kinetin的Kao培养基中诱导愈伤组织。将培养获得的936个愈伤组织转到3种不定芽诱导培养基MS + 5 mg ? L-1 zeatin + 2 mg · L^-1 IAA;MS + 2 mg · L^-1 zeatin + 2 mg · L^-1 IAA;MS + 0.5 mg · L^-1 6-BA + 0.5 mg · L^-1 NAA上诱导芽分化,当芽长3 cm左右时,转到1/2 MS + 0.2 mg · L^-1 NAA的生根培养基上诱导生根,共获得了96株再生植株,对其进行形态学、细胞学和分子生物学鉴定。形态学观察显示再生植株表型变化大,包括3个亲本的中间型或两个亲本的中间型;PCR技术鉴定结果显示96株杂种植株中93株为细胞质雄性不育特性;染色体计数显示,再生的植株染色体数变化范围广（2n = 38 ~ 64）,但未发现染色体数总和与3种融合亲本染色体数总数（2n = 74）相一致的个体。基因组原位杂交（Genomic in situ hybridization,GISH）和扩增片段长度多态性（Amplified fragment length polymorphism,AFLP）分析结果表明,再生植株基因组中分别检测出大白菜、青花菜、叶用芥菜的DNA片段,证实了大白菜、青花菜、叶用芥菜种间体细胞杂交种的真实性。     

花椰菜—黑芥体细胞杂交获得抗黑腐病异附加系新材料

 以花椰菜—黑芥体细胞杂种的自交及回交后代为材料,建立了结合抗病性人工接种鉴定,流式细胞仪DNA含量分析,B基因组特异分子标记和染色体原位杂交的大规模群体分析技术,在50份高代自交及回交材料中筛选获得了20份形态学特征偏向花椰菜,30份呈花椰菜—黑芥中间类型的材料。细胞DNA含量检测（FLC）及染色体计数结果表明,偏花椰菜的类型,DNA含量基本在2.24 pg · cell^-1以下,染色体数在28条以下。经连续3年黑腐病病菌人工接种鉴定,获得高抗株系12个,抗性株系17个,占全部受试材料的14%。对其中3个（PFCN29 BC3-3-5、PFCN16-1 S2BC3-107和PFCN14-1 S1BC4-123）形态偏花椰菜且高抗黑腐病的植株进行基因组原位杂交,结果显示其分别为花椰菜染色体组附加了2、7和8条黑芥染色体的异附加系材料。这些植株可以用于后续的研究和作为育种桥梁材料,进一步获得黑腐病抗性的单体附加系或渐渗系。本研究表明通过体细胞杂交转育野生优异抗性资源,创新甘蓝类蔬菜育种种质的可行性。     

采用同核体单孢杂交育种技术选育双孢蘑菇

采用同核体单孢杂交育种技术,以低温高产的双1菌株和高温低产的双5菌株为亲本,选育双孢蘑菇优良新品种.通过担孢子分离和同核体的鉴别,获得同核体菌株共20个,同核体间配对杂交组合99个,获得10个杂交异核体菌株;经过菇房、出菇地棚栽培和出菇大棚生产性试验,筛选出高产优质杂交菌株Z1.     

Cuticular hydrocarbons in two parapatric species of ants and their hybrid

Discrimination between nestmates and non‐nestmates in social insects is thought to rely on the pattern of cuticular hydrocarbons. We investigated the cuticular hydrocarbon profiles of 2 parapatric sibling ant species, Temnothorax nylanderi (Förster, 1850) and Temnothorax crassispinus (Karavaiev, 1926), and their hybrid. We found that although the profiles show considerable similarities, a discriminant analysis based on the relative peak areas of cuticular hydrocarbons separates the 3 taxa. The profiles of hybrids were not consistently intermediate between those of the paternal species, suggesting either non‐additive interactions among the parental biosynthetic pathways or systematic differences in environment‐derived odor cues.     

辣椒细胞质雄性不育系和保持系SSH-cDNA文库的构建

以辣椒细胞质雄性不育系A1、A5为驱动方(driver),以其相应的保持系B1、B5为测验方(tester),利用抑制差减杂交技术构建了1个辣椒细胞质雄性不育保持系表达基因的正向差减cDNA文库。从差减文库中筛选到189个阳性单克隆,经高密度点阵膜杂交筛选和测序分析,共获得了42条表达序列标签(ESTs)。BLAST比对分析后,得到了35条有比对结果的序列,其中包括26条已知功能基因序列和9条未知功能基因序列,已知基因功能多与基础物质代谢、胁迫应答、信号转导等方面有关;有7条ESTs没有比对结果,可能代表一些新基因。     

水稻野栽杂交受粉子房离体培养研究

对野生稻与栽培稻杂交受粉子房离体培养的试验结果表明，不同杂交配组类型的培养效果为：栽×栽（同一品种）＞栽×栽（不同品种）＞栽×野＞野×栽＞野×野。１６个水稻野栽杂交组合的结实率为０％～４７．４％。受粉子房离体培养时间以授粉２～３ｄ龄较为适宜。离体培养的水稻杂交受粉子房的发育有三种情况，即子房（胚胎）滞育或败育、结实和形成植株。胚继续培养有利于胚存活和发育与植株产生。在诱导产生的３８个植株中，未见白化苗     

Variability of communities and physiological characteristics between free-living bacteria and attached bacteria during the PAH biodegradation in a soil/water system

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) via free-living and attached micro-organisms in soil/water systems was observed in order to examine the variability in the community dynamics and physiological profiles of the micro-organisms. As determined by fluorescence in situ hybridization (FISH), the Domain Bacteria, consisting of three phyla α-, β- and γ-Proteobacteria, reached 41.27–56.05% of all organisms in the soil/water system for PAH biodegradation. Among the free-living species, Proteobacteria, including Brevundimonas (Pseudomonas) diminuta, Caulobacter spp., Mycoplana bullata, Acidovorax spp. and Pseudomonas aeruginosa were found to be dominant—making up 93.51–99.80% of the population—and therefore seem to be associated with PAH biodegradation. Total plate count numbers and the count of Pseudomonas sp. present in the free-living population increased to between 10³ and 10⁶ CFU ml⁻¹ when clay with very low organic matter content was used as the matrix for PAH degradation. However, total plate count microbial numbers increased to only 10¹–10² CFU ml⁻¹ using natural soil from Taichung containing 1.883% organic matter. The soil organic content (SOM) seemed to affect the mass transfer of PAH in soil, leading to the difference in PAH biodegradation. Two different approaches, which included community-level physiological profiling (CLPP) and ectoenzymatic activities, were used to explain the functional diversity between free-living and attached bacteria. The free-living and attached bacterial communities from the clay system showed proportionately greater differences using CLPP. Relatively high levels of esterases, aminopeptidases and some specific glycolysis-gluconeogenesis enzymes gave an identifiable correlation with PAH biodegradation. The differences in bacterial composition, numbers and physiological characteristics show that free-living and attached micro-organisms may play different biochemical roles in PAH degradation in soil.     

Frankia共生固氮基因的分离与克隆

利用ＤＮＡ－ＤＮＡ杂交方法，分离细枝木麻黄的Ｆｒａｎｋｉａ菌株Ｃｏ０１的ｎｉｆ克隆ｐＣｃ１ＧＸ，赤杨内生菌株Ａｔ４的ｎｉｆ克隆ｐＡｔ１ＧＸ及沙棘的ＦｒａｎｋｉａＨｒ１８的ｎｉｆ克隆ｐＨｒ１８ＧＸ、ｐＨｒ１１－③ＧＸ。用基因功能互补法，从Ｆｒａｎｋｉａ菌株Ａｔ４的基因文库中分离到二个可能可互补豌豆根瘤菌ｎｏｄ基因功能的克隆ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ，并制作了ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ的亚克隆，予进一步实验，获得充分的证据证明ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ是否带有Ｆｒａｎｋｉａ菌株Ａｔ４的ｎｏｄ基因．     

Evaluation of fluorochromes for imaging bacteria in soil

The distribution of inoculated Escherichia coli cells and indigenous bacteria in a silt loam soil and a sandy soil was studied using fluorescence microscopy techniques. Ethidium bromide stained inoculated cells against the soil and resin background. Satisfactory results were also obtained with epifluorescence microscopy of samples stained with calcofluor white M2R (CFW) and 4′,6-diamidino-2-phenylindole (DAPI). Indigenous soil bacteria were visualized in soil thin sections, after staining with ethidium bromide, using fluorescence microscopy. The density of these bacteria was estimated to be 10⁷-10⁸ cells/cm³. Inoculated E. coli cells, stained with one of the green fluorochromes (fluorescein isothiocyanate (FITC), 5-(4,6-dichlorotriazinyl)aminofluorescein, or eosin Y), could be clearly distinguished in sandy soil thin sections. Confocal laser scanning microscopy with 3D image reconstruction was also successfully applied to characterize distribution of E. coli introduced to soil. The fate of introduced bacteria and the location of indigenous bacteria in soil can be confirmed using the microscopic techniques described in this paper.     

基因芯片技术检测5种马病毒

通过分子克隆技术获得马疱疹病毒1型(Equineherpesvirus1,EHV1)、马动脉炎病毒(Equinearteritisvirus,EAV)、马流感病毒(Equineinfluenzavirus,EIV)、马传染性贫血病毒(Equineinfectiousanaemiavirus,EIAV)和东部马脑脊髓炎病毒(East-ernequineencephalomyelitisvirus,EEEV)等5种病毒各一段高度保守的特异性基因片段,用芯片点样仪逐点分配到处理过的玻片上,制备成检测芯片。提取样品中的RNA,进行反转录和荧光标记后滴加到芯片上进行特异性杂交,对杂交结果进行扫描检测和计算机软件分析。结果显示,制备的基因芯片可同时检测和鉴别上述5种病毒,可检测到阳性杂交信号的最高稀释度为10-6的病毒液,约25个病毒DNA拷贝,但其它病毒材料未见红色荧光信号,证明了本方法的特异性。在进口马的隔离检疫期间,采集马鼻肺炎、马动脉炎中和抗体阳性但病毒分离阴性马匹的白细胞悬液,分别在EHV1和EAV位点处可检测到阳性杂交信号。证明基因芯片技术不但快速、准确和敏感,而且可同时进行多种病毒的检测。