小麦杂交坏死蛋白质组学研究

小麦杂交坏死是某些小麦杂交种表现出的叶片提前逐渐死亡的现象。它是由两个坏死基因Ne1和Ne2在杂交种中相遇后发生显性互补引起的。坏死从叶片尖端逐渐过渡到叶片基部，从成熟叶片发展到幼嫩叶片。一些严重坏死的F1完成它的生活周期前就在不同的生长阶段死去，无法获得F1种子，这就限制了携带优良性状的亲本的选择和优良基因的交流。另外，小麦杂交坏死是一个独特的研究植物程序性死亡的遗传系统。虽然小麦杂交坏死这种现象已经发现很多年，但其详细的分子机理却仍然未知。对小麦杂交坏死的分子机理进行深入研究将有助于克服小麦杂交利用中杂交坏死的遗传障碍，此外，也为深入研究植物的PCD机理提供可操作靶分子。 本论文采用高通量蛋白质组研究技术对小麦杂交坏死进行了研究。携带坏死基因Ne2的小麦品种Pan555（P）和携带坏死基因Ne2的小麦品种Zheng891（Z）生长发育完全正常，将两个亲本杂交，所得杂交F1代PZF1表现杂交坏死。在小麦生长阶段8，旗叶（Flag leaf）刚刚出现，PZF1的旗叶下第一片叶子（FL-1）还是完全绿色，FL-2叶尖开始有坏死斑出现。在这个阶段，分别将PZF1，P，Z的FL-2叶剪成相等的尖，中，基三段。我们选择的PZF1的FL-2叶，其叶尖段已经有成片的坏死斑出现;中间段零星出现少量坏死斑点;基部段和亲本一样还是完全的绿色，代表坏死进程中的不同阶段。又选PZF1的FL-1和FL-2分别代表杂交坏死启动前和杂交坏死启动后。两个亲本P和Z的FL-2叶的三段及FL-1叶正常，都是完全绿色。 首先分别分析了PZF1，P和Z的FL-2叶的尖、中、基三段的蛋白表达情况。在PZF1的尖、中、基三段共检测到23个差异表达蛋白点。这23个点在两个亲本的尖、中、基三段中的表达丰度没有显著差异（p<0.05），说明这23个蛋白的差异表达不是由于叶段的不同引起，确与杂交坏死相关。对这23个蛋白进行了MALDI-TOF质谱鉴定，其中18个得到成功鉴定。然后对PZF1，P和Z的FL-1叶和FL-2叶的蛋白表达情况进行了分析。与PZF1的FL-1叶比较，在FL-2叶中检测到19个蛋白上调，20个蛋白下调。这39个蛋白的丰度在两个亲本的FL-1和FL-2叶之间没有显著差异，说明这39个蛋白的差异表达不是由于叶位的不同引起，确与杂交坏死相关。对这39个蛋白进行质谱鉴定其中26个得到成功鉴定。 根据被鉴定蛋白的功能及其表达丰度的变化，对这些蛋白在小麦杂交坏死中可能的作用进行了讨论。与PZF1的FL-2叶基部相比，S-腺苷同型半胱氨酸水解酶（S-adenosyl homocysteine hydrolase）在中部极显著（p<0.01）下调，而在中部和尖段之间没有显著差异，保持低丰度不变。腺苷甲硫氨酸3（AdoMet synthase 3）和甲硫氨酸合成酶1（Methionine synthase 1）都在PZF1的FL-2叶尖段上调。甲基化循环中的这3个酶比例的不协调可能会以不同的方式加速细胞老化。 与PZF1的FL-1叶比较，尿卟啉环脱羧酶（Uroporphyrinogen decarboxylase）在FL-2叶中下调，这将引起尿卟啉环III的积累。脂加氧酶（Lipoxygenases）在FL-2叶中上调。尿卟啉环III的积累和脂加氧酶的上调都会引起细胞内活性氧的增加。另外活性氧和脂加氧酶都会使脂发生过氧化作用，进而导致细胞膜完整性受到破坏，最终可能导致细胞死亡。 与基部段比较，在PZF1的FL-2叶的尖段和/或中间段;以及与PZF1的FL-1叶比较，在FL-2叶中，都有很多防御性蛋白的上调，这暗示应对活性氧、脂过氧化、甲基化循环中三个酶比例的不协调等引起的对细胞的破坏作用，细胞可能启动了抗细胞死亡系统来应对这种细胞内部的胁迫。 然而，与基部段比较，一些能量相关蛋白在PZF1的FL-2叶的尖段和/或中间段;以及与PZF1的FL-1叶比较，在FL-2叶中的异常表达可能会以干扰能量循环的方式加速细胞死亡。另外，与FL-2基部段比较，在尖段和/或中间段，以及与PZF1的FL-1比较，在FL-2中，都有一些防御性蛋白、蛋白合成相关的蛋白以及单链DNA结合蛋白下调，它们的变化可能会降低细胞的抵抗力，蛋白合成能力以及DNA修复能力。细胞正常代谢的很多方面都受到干扰从而使PZF1叶细胞最终走向死亡。 本研究中发现了三个甲基化循环中的酶变化，而且S-腺苷同型半胱氨酸水解酶是在坏死进程的较早阶段发生下调，它的变化可能是小麦杂交坏死的一个诱因，这暗示小麦杂交坏死可能是一个表观遗传学事件。另外本研究还发现一些和活性氧，脂氧化等相关的蛋白的变化，而活性氧增加和脂氧化都是细胞凋亡的典型特征。所以本研究为表观遗传细胞凋亡和氧化胁迫细胞凋亡的研究提供了很有价值的信息。

葡萄杂种实生苗糖酸品质童期鉴定的初步研究

对山葡萄(Vitis amurensis)杂交的F1、F2、F3杂种后代中，果实搪、酸含量不同的实生苗叶片，进行了Vc、VB1Vpp的分析测定，并对这些植株的叶片、卷须、一年生技条扦插芽和种子播出的幼苗分别进行了过氧化物酶同功酶的聚丙烯酰胺(PO-lyacrylamide)垂直平板电泳分析。实脸结果表明:在三代的实生苗中，Vc、VB1、Vpp的含量与含糖量均呈正相关，Vc和Vpp与含酸量负相关，VB1与含酸量呈正相关，Vc与Vpp呈正相关.Vc含量低的实生苗易受冻而导致萌芽推迟，Vc在果实潜色期间呈动态变化，着色期早的Vc含量降低，正处在着色期的Vc含量最低，着色晚的Vc含量就高。叶片中Vc、Vpp含量可作为实生苗含量筛选的参考指标。 而且发现，在实生苗幼龄阶段，通过过氧化物酶同功酶能够从品质上进行单株分离与筛选。一年生枝条的扦插芽以及生长季节的卷须和叶片为材料的分离筛选效果相对稍差。F1实生苗中，抗寒、含糖量高的单株表达出双亲的酶带;F2品贡优良的单株从表达出亲本中含糖童截的酶潜并且酶带数目相对少些的单株中产生;F3品质优良的单株则从酶谱复杂、杂种谱带较多的单株产生;品质性状较劣，同功酶表现出相反的结果。这为育种工作中果实品质性状的童期鉴定提供了参考依据。在实际鉴定工作上，最好有几种方法，几种酶的功酶联合使用。

Effect of low cost feed on the production of walking catfish, Clarias batrachus in farmer's ponds

A feeding trial was conducted for six months in farmer's ponds to assess the performance of BFRI formulated catfish feed on the growth and survival of Clarias batrachus (L.). Nine interested farmers and their ponds (size range: 10-15 dec) in the Barera union of Mymensingh Sadar were selected. The ponds were divided into 3 treatments each with 3 replications. Among the three treatment diets, two diets - traditional (F1) and BFRI formulated (F3) were prepared by using low cost agro-based locally available ingredients and the commercial diets was Saudi-Bangla Grower-1 (F2). The diets were designed as F1, F2 and F3 for traditional (20.40% protein), Commercial (31% protein) and BFRI formulated (30.44% protein) diets respectively. The fingerlings of catfish (7.3 g) were collected from local fish vendors and stocked at the rate of 100/dec. Feeding rates were adjusted by weight after fortnightly sampling of fish. Feeding rate were 10 and 8% of the total body weight respectively for 1st, 2nd month and 5% for the rest of the experimental period. The range of some selected water quality parameters were as follows: dissolved oxygen 4.0 - 7.4 mg/l, temperature 24.0°- 33.9°C, pH 6.8 - 8.00, and transparency 17.0 - 32.00 cm. Which showed suitability of the ponds for rearing fish. At the end of the experiment, significantly highest gain (p<0.05) in weight (1210.96% ±87) and lowest gain in weight (865.25% ±90) were observed in the group of fish fed on diets F3 and F1 respectively. However, no significant differences in growth (p>0.05) was observed in fish fed on commercial diet (F2) and BFRI formulated diet (F3). The FCR value ranged between 2.00 and 2.80 with the traditional diet (F1) showing significantly lower FCR. The total production of fish ranged between 1398.08 and 2145.34 kg/ha with F3 diet resulting in the highest production and net profit. A simple economic analysis showed that fish fed with BFRI formulated (F3) diet resulted in the highest net profit in farmer's pond.

饲养层FGF2表达量对猕猴胚胎干细胞自我更新及多能性的影响

用转基因和RNA干扰(RNAi)法建立5组不同成纤维细胞生长因子-2(fibroblast growth factor-2,FGF2)表达量的猕猴耳部皮肤成纤维细胞(MESF)系:过表达FGF2组(f1),过表达的阴性对照组(f2),FGF2 RNA干扰组(f3),RNA干扰的阴性对照组(f4)和未作任何处理的对照组(f5).5组MESF的FGF2表达量相对值为f1:f2:f3:f4:f5=4:2:1:2:2;c-fos,TGF-β1,INHBA,Gremlinl在f1中表达量上升,在f3中表达量下降;BMP4,TGF-β2在f1中表达量下降,在f3中表达量上升;表明内源FGF2能够作用于MESF的TGF-β信号通路,引起相关基因表达量的变化.用这砦细胞作为饲养层长期培养(10代)猕猴胚胎干细胞(RhESC),结果在f1上培养的RhESC增殖速度都比对照组快,并且c-fos,TGF-β1,INHBA,Gremlinl,Oct-4,Nanog,Sox2表达量均上升,BMP4表达下调;在f3上培养的RhESC增殖较慢,BMP4表达上调,c-fos,TGF-β1,INHBA,Gremlinl,Oct-4,Nanog,Sox2表达下调.5组MESF上培养的RhESC形成的EB均表达各胚层早期标记基因(marker),说明RhESC的多能性没有受到影响,但表达量有差异,f1上RhESC形成的EB所有marker都低表达.结果表明饲养层的FGF2含量不仅影响自身相关基凶的表达,还对RhESC的自我更新有一定的作用.

云南纳帕海越冬黑颈鹤日间行为模式与年龄和集群的关系

2006年10月2007年5月,在云南省西北部纳帕海,采用路线调查结合瞬时扫描行为取样法,对越冬黑颈鹤(Grus nigricollis)种群的时间分配及其与年龄、集群和时间的关系进行了观察.结果说明,黑颈鹤越冬活动主要足以觅食为主,占日间时间的(76.81±9.1)%.越冬期间黑颈鹤日间行为的节律性较为明显,具有适应高寒气候的特点.集群形式对成鹤的行为有着显著影响,集群和家庭中活动的成鹤红觅食、警戒和争斗中存在显著差异(F1.76=0.27、0.77,U=2779,P=0.001-0.000).年龄是影响鹤群行为的因素之一.幼鹤相比成鹤有较多的觅食时间和休息时间,警戒行为比例较低(F1.76=0.04-2.59,U=188-2779,P=0.006-0.000),且小受集群形式的影响.随着越冬期间的早、中、晚3个时期的环境变化,黑颁鹤的时间分配有显著变化(F2.36=4.63-26.54,x22=5.29-13.68,P=0.0016-0.000).不同越冬地的黑颈鹤行为存在差异.气候、食物资源和人为影响可能是造成这些差异的主要因素.

不同世代改良型路南奶山羊血液免疫学参数的比较研究

本文测定了“改良型路南奶山羊”的亲本及改良不同世代 (F1,F2 ,F3,F4 )母羊的外周血T淋巴细胞转化率、ANAE阳性率、B淋巴细胞ZYC花环率、红细胞IC花环率、C3b花环率以及血清γ -球蛋白等免疫学参数 ,比较研究其随着世代增递的变异情况。结果表明 :从亲代到F4代随着改良世代的递增 ,T淋巴细胞转化率、ANAE阳性率、红细胞C3b花环率和IC花环率和血清γ -球蛋白含量均呈F1>F2 >P >F3>F4变化 ,ZYC花环率呈F1>P >F2 >F3>F4变化。回归分析的结果表明 :从亲代到改良四代 ,上述各项免疫学参数均呈显著负相关 ,趋势线为下降趋势。即随着世代的递增有逐代递减的趋势。结果提示 :用西农莎能羊改良路南圭山羊 ,其改良一代和二代的上述各项血液免疫学参数均略高于路南圭山羊 ,以后随着世代的递增有逐代递减的趋势。预示着随着世代的递增 ,改良型路南奶山羊的体质状况 ,免疫功能和抗病能力可能会出现下降趋势。

东北梅花鹿与东北马鹿种间杂交F_(1)能育性的细胞遗传学基础的研究

从F_(1)的染色体组型、G带、C带和Ag-NORs观察表明: F_(1)双亲鹿 的染色体高度同源, 差异只涉及一个罗伯逊易位, F1两性皆育说明其亲本鹿之间 隔离机制不完善。这两种鹿应属同一个孟德尔群体。图版3参11

两个家系猪的DNA指纹图谱遗传分析

用人源小卫星探针33.6和33.15获得了两个家系猪的DNA指纹图谱。通过对F1和F2代家系分析,证实DNA指纹图带以孟德尔方式遗传。在家系2(探针33.6)的1个后代中发现了一条新突变带。文中还对这两个探针检测到的位点数进行了估测。

Gene mapping of a new coat mutation in mouse

Gene mapping of a mouse coat mutation has been investigated. First, 100 10-bp random primers were used to amplify DNA, but the mutation could not be located by this method because there were no correlation between the amplified products and coat phenotypes. Second, by using Idh1, Car2, Mup1, Pgb1, Hbb, Es10, Es1, Mod1, Gdc1, Ce2, Es3 as genetic markers, linkage test crosses (two-point test) consisting of intercrossing uncovered BALB/c mice (homozygotes) to CBA/N and C57BL/6 mice with normal hair and backcrossing the heterozygotes of the F1 to the uncovered BALB/c mice were made. It was soon evident that the mutation was linked to Es3 on chromosome 11. Furthermore, three-point test was made by using Es3 and D11Mit8 (a microsatellite DNA) as genetic markers. The result showed that the mutation was linked to Es3 with the percentage recombination of (7.89 +/- 2.19)%, and linked to D11Mit8 with the percentage recombination of (26.38 +/- 3.57)%. The percentage recombination between Es3 and D11Mit8 was (32.90 +/- 3.81)%. The mutation was named Uncovered, with the symbol Uncv. According to the recombinations, the loci order was D11Mit8-26.30 +/- 3.57- Uncv-7.89 +/- 2.19-Es3. From the location on the chromosome, it was concluded that the mutation was a new mutation which affected the skin and hair structure of mouse. The Uncv has entered MGD (Mouse Genome Database).

红白锦鲤人工雌核发育纯系的微卫星标记分析

利用连续两代人工雌核发育操作 ,构建了极具观赏价值的红白锦鲤人工雌核发育系。应用 6对微卫星引物进行遗传标记研究 ,检测分析了该雌核发育系内个体间的遗传同质性及其基因座位的纯合度。实验结果显示 ,11尾经人工挑选具有相同表形、极具观赏价值的F1代性成熟个体的微卫星扩增图谱呈现出高度的一致性。同时 ,随机挑选的人工雌核发育F2代个体在所检测的基因座位均呈现纯合 ,扩增图谱呈现了高度的一致 ,从分子水平上证实了本实验所获得的红白人工雌核发育F2代是一个纯系

转基因异源四倍体鲫鲤F_1的研究

采用显微注射法将含有鲤鱼 β actin基因启动子的草鱼生长激素基因“全鱼”基因pCAgcGHc转入异源四倍体鲫鲤 ,然后使其自交得到转基因异源四倍体鲫鲤F1,对 15 0日龄F1体重和体长进行检测 ,可明显看见转基因异源四倍体鲫鲤F1的生长优势 ;取F12 0尾 ,提取尾鳍基因组DNA ,采用合适的引物 ,PCR方法检测转基因异源四倍体鲫鲤F1是否含有外源生长激素基因 ,结果 15 0日龄F1阳性率达到 90 % ,且有些雄性个体可以挤出少量精液 ,而普通 15 0日龄异源四倍体鲫鲤无此现象。文章阐明了

转“全鱼”生长激素基因鲤鱼及其F_1遗传分析

采用显微注射方法, 研制出转"全鱼"生长激素基因鲤鱼. 比较分析了转基因鲤鱼(P0)获得性状多样性, 筛选获得具有显著快速生长效应的转基因鱼个体; 繁殖力参数比较发现, 快速生长转基因鱼的繁殖能力无实质改变. 对F1转基因鱼转植基因分离和性状分布进行了遗传分析, 结果证实转植基因在2, 3条染色体上整合, 转植基因不同整合位点的生物学效应存在差异, 这为快速生长转基因鱼育种奠定了基础.

人生长激素和转移的人生长激素基因对去垂体鱼的生长代偿效应

通过显微注射技术,将小鼠重金属螯合蛋白(MT-1)基因启动顺序与人生长激素基因顺序的重组体pMThGH注入鲤鱼(Cyprinus carpio)的受精卵内,由此发育的转基因鱼及其后代F1和F2均显示出快速生长效应。去垂体后,转基因鲤鱼F2持续生长,而非转基因鲤鱼和鲫鱼(Carassius auratus)的生长停止。给去垂体的鲫鱼腹腔注射生物合成的人生长激素(hGH),可恢复其生长。实验结果表明,转基因鱼体内表达和体外生物合成的hGH均能代偿鲤鱼和鲫鱼的内源生长激素并刺激去垂体鱼的生长。

Enhanced grass carp reovirus resistance of Mx-transgenic rare minnow (Gobiocypris rarus)

In the interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins have direct antiviral activity and inhibit a wide range of viruses by blocking an early stage of the viral genome replication cycle. However, antiviral activity of piscine Mx remains unclear in vivo. In the present study, an Mx-like gene was cloned, characterized and gene-transferred in rare minnow Gobiocypris rarus, and its antiviral activity was confirmed in vivo. The full length of the rare minnow Mx-like cDNA is 2241 bp in length and encodes a polypeptide of 625 amino acids with an estimated molecular mass of 70.928 kDa and a predicted isoelectric point of 7.33. Analysis of the deduced amino acid sequence indicated that the mature peptide contains an amino-terminal tripartite GTP-binding motif, a dynamin family signature sequence, a GTPase effector domain and two carboxy-terminal leucine zipper motifs, and is the most similar to the crucian carp (Carassius auratus) Mx3 sequence with an identity of 89%. Both P0 and F1 generations of Mx-transgenic rare minnow demonstrated very significantly high survival rate to GCRV infection (P < 0.01). The mRNA expression of Mx gene was consistent with survival rate in F1 generation. The virus yield was also concurrent with survival time using electron microscope technology. Rare minnow has Mx gene(s) of its own but introducing more Mx gene improves their resistance to GCRV. Mx-transgenic rare minnow might contribute to control the GCRV diseases. (C) 2008 Published by Elsevier Ltd.

Partial and total replacement of fishmeal with poultry by-product meal in diets for gibel carp, Carassius auratus gibelio Bloch

Triplicate groups of gibel carp Carassius auratus gibelio Bloch (initial body weight: 4.89 g) were fed for 8 weeks at 24.8-30.8 degrees C with nine isonitrogenous and isoenergetic diets. The control diet (F1) used white fishmeal (FM) as the sole protein source. In the other eight diets (F2-F9), 40.5-100% of FM protein was substituted by poultry by-product meal (PBM) at 8.5% increments. The specific growth rate (SGR), feed efficiency ratio, protein efficiency ratio, protein retention efficiency and energy retention rate for fish fed PBM diets (F2-F9) were all higher, but not always significantly, than those for fish fed F1. All apparent digestibility coefficients for fish fed PBM diets were lower than those for fish fed F1. Fish fed F1 had a significantly higher hepatosomatic index value than fish fed PBM diets (P < 0.05). No significant (P > 0.05) effect of diet was found in whole-body moisture and fat content. Whole-body protein and energy content for fish fed PBM diets were slightly higher than that for fish fed F1. The optimal replacement level of FM by PBM was estimated by second-order polynomial regression to be 66.5% in protein.