转基因异源四倍体鲫鲤F1的研究

采用显微注射法将含有鲁鱼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150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F120尾,提取尾鲤基因组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150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150日龄异源四倍体鲫鲤无此现象,文章阐明了通过近交筛选转基因异源四倍体鲫鲤种系具有重要意义。     

转几丁质酶和葡聚糖酶基因水稻对土壤三种弹尾目昆虫和一种环节动物的影响

采用 3个试验研究转几丁质酶和苜蓿 1,3- β -葡聚糖酶双价基因水稻对土壤弹尾目昆虫和环节动物线蚓的影响。①平皿实验 :观察弹尾目与转基因水稻组织间的营养关系及其在无土壤情况下对弹尾目昆虫繁殖率的短期影响 ;②土壤 -植物残留物单物种微宇宙试验 :观察土壤中不同转基因水稻组织含量对土壤动物繁殖率的短期影响 ;③土壤 -植物残留物多物种微宇宙试验 :模拟大田状况 ,观察多物种共存较长时间后转基因水稻对土壤动物密度的影响。结果表明 :Folsomiacandida取食新鲜转基因水稻叶片和根组织 2 1d后 ,繁殖率比野生型水稻组分别降低 4 5 1% (F1,8=9 9,P <0 0 5 )和 30 5 % (F1,8=5 8,P <0 0 5 ) ;在掺有转基因水稻组织 (30g/kg)的土壤中 ,F .candida 2 1d后的繁殖率比野生型水稻组下降 19 3% (F3 ,16=3 6 ,P <0 0 5 ) ;在掺有转基因水稻组织土壤 (30g/kg)的多物种实验中 ,转基因水稻组中的F .candida、Enchytraeuscrypticus16周后的密度比野生型水稻组分别降低 19 4 % (F1,48=6 8,P <0 0 5 )和 38 0 % (F1,48=6 4 2 ,P <0 0 0 1) ,而Sinellacurviseta的密度升高 84 8% (F1,48=6 4 2 ,P <0 0 0 1)。因此 ,转几丁质酶和葡聚糖酶双价基因水稻对土壤弹尾目和线蚓都是有影响的 ,对某些种类表现     

马氏珠母贝雌雄同体和自体受精的研究

对马氏珠母贝(Pinctada martensiiDunker)成体的性腺进行外观和切片观察,可把马氏珠母贝雌雄同体的个体分为2种类型,其中A类型可以从性腺外观特征上辨别,而B类型需性腺穿刺或组织切片来确认。对试验群体的性腺的类型进行的初步统计表明,雌雄同体个体比例A类型为4.6%,B类型为3.4%,其中雌雄生殖细胞均接近成熟的个体分别约为1.8%和2.1%。通过解剖、0.07‰的氨海水处理,对雌雄同体个体的自体授精及其早期胚胎发育进行了研究,发现自体授精的受精率低于异体授精,同时在发育过程中有较多的卵裂球解体,但仍有部分胚胎可以发育成D型幼虫。     

哮喘大鼠肺泡巨噬细胞蛋白激酶C-α表达及丹参对其影响

探讨蛋白激酶 C (Protein Kinase-α,PKCα)在大鼠哮喘模型肺泡巨噬细胞中的表达及丹参的治疗作用。哮喘模型大鼠分为四组。 A组正常对照 ;B组即刻激发组 ;C组哮喘发作一周组 ;D组丹参治疗组。每组分别测定肺功能 ,支气管肺泡灌洗液 (BAL F) ,分离巨噬细胞作免疫细胞化学染色 ,观察 PKCα在大鼠哮喘模型肺泡巨噬细胞中的表达及丹参对其影响。结果发现 A组中肺泡巨噬细胞见 PKCα少量表达 ,而 B、 C、 D组则见大量巨噬细胞 PKCα表达 ,但 D组巨噬细胞 PKCα表达比例显著低于 B、 C组 (P<0 .0 1 )。四组肺泡巨噬细胞 PKCα表达百分比分别为 A组 3.49± 2 .40 % ,B组 85 .0 6± 5 .46 % ,C组 90 .2 3± 2 .87% ,D组 37.89± 7.2 1 %。且 A、 B、 C组肺泡巨噬细胞 PKCα表达比例与气道阻力呈正相关 (n=2 1、 r=0 .74　 P<0 .0 1 ) ,结果说明丹参对哮喘大鼠 BAL F中巨噬细胞 PKCα表达有抑制作用 ,提示肺泡巨噬细胞 PKCα表达参与哮喘发病过程 ,而丹参则对这一过程有抑制作用。     

携带HLA-B2704基因转基因小鼠技术的建立

应用显微注射法制备携带HLA B2 70 4基因的转基因小鼠 .对 2 86只昆明小鼠激素注射进行超排卵 ,采集受精卵 ,将含HLA B2 70 4基因的基因组DNA片段 (简称HLA B2 70 4DNA)显微注射到受精卵原核内 ,把注射存活的两细胞期受精卵移入假孕鼠的输卵管内使其发育产生后代 .用PCR方法进行F0代仔鼠及F1代仔鼠的转基因整合的检测 .利用RT PCR检测阳性鼠中的HLA B2 70 4转基因的表达 .采集了 84 11个卵 ,可注射卵 6 6 0 9个 ,其中注射存活的两细胞期受精卵 4 2 77个 ,卵的注射存活率为 6 4 7%.将卵移入 15 3只假孕鼠 ,其中 2 6只怀孕产仔 ,存活 10 1只 .在 10只F0代仔鼠基因组中有HLA B2 70 4基因整合 ,整合率为 9 9%.转基因阳性鼠F0代之间以及与正常鼠之间进行交配 ,产生的F1代仔鼠 78只 ,其中 15只为阳性 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有HLA B2 70 4转基因mRNA的表达 .在HLA B2 70 4转基因阳性小鼠中 ,6只小鼠皮肤出现脱毛 ,1只小鼠的足部及足趾明显红肿 ,2只在脱毛同时明显畏光 ,1只出现腹泻 .结果表明 ,成功地建立了HLA B2 70 4的转基因小鼠技术 ,该小鼠类似强直性脊柱炎的小鼠模型 .     

籼稻转反义蜡质基因后代的直链淀粉含量测定和纯系选育

通过根癌农杆菌介导将反义蜡质基因导入籼型雄性不育保持系龙特甫B中,获得30个PCR检测为阳性的转基因植株,其中,28个为Southern检测阳性.T1种子直链淀粉含量测定结果表明,有21个转基因植株比龙特甫B明显下降,下降幅度为3%-13%,并在部分转基因植株的种子中观察到蜡质状籽粒;对6个转基因植株进行了不同世代的直链淀粉含量测定,在L3和L5的T4代中,选择到直链淀粉含量分别为15.9%和8.4%的纯合株系,经凝胶电泳测定,Wx蛋白量明显降低,并与直链淀粉含量下降表现一致.以L3-1-1-1(15,9%)和L5-8-2-1(8.4%)纯合株系为亲本,分别与龙特甫A进行成对杂交和回交,并测定了F1和B1F1种子直链淀粉含量,以L3-1-1-1作亲本的F1为21.4%,B1F1为17.1%;以L5-8-2-1作亲本的F1为13.6%,B1F1为9.3%,结果表明在不育系的转育过程中,以中低直链淀粉含量的转基因纯合株系为亲本,能有效降低F1和B1F1的种子直链淀粉含量.     

人SR-AI转基因小鼠遗传与繁育的研究

目的探讨被转入的人SR-AI基因的整合与复制情况及其对小鼠繁育的影响.方法采用类似系祖建系的方法,对人SR-AI转基因小鼠的2、3534、3560、3638、3639五个系列进行了繁育.并用PCR和Southermblot的方法,检测五个系列小鼠尾组织的DNA样品.结果五个系列小鼠共产仔431只,PCR检出阳性小鼠178只,阳性率为41.2%.在3639系的F1、F2、F3代及纯合子转基因鼠中PCR阳性率分别为47.8%、71.3%、75%和100%.结论人SR-AI基因在子代鼠中能稳定遗传,且对生殖和发育无明显影响.     

转青鱼生长激素基因异源四倍体鲫鲤

生态安全性是转基因鱼走向市场的瓶颈,通过转基因四倍体鱼同转基因二倍体鱼杂交获得不育的转基因三倍体鱼是解决该问题的有效途径之一.本研究构建了青鱼β-actin基因启动子和青鱼生长激素（GH）基因精确连接的＂全鱼＂基因pbcAbcGHc;并采用显微注射法将pbcAbcGHc导入异源四倍体鲫鲤受精卵.对照养殖结果表明,150日龄的转基因异源四倍体鲫鲤原代（P0）的体重及体长明显大于对照组.选择60尾P0代转基因异源四倍体鲫鲤,采用PCR方法检测出外源青鱼GH基因在P0代转基因四倍体尾鳍基因组DNA中的整合率为90%;对20尾雄性P0代转基因四倍体精液样本的PCR检测发现,13个样本具有外源青鱼GH基因的整合.在一尾生长速度显著的P0代转基因四倍体鲫鲤的肌肉、肝脏、肾脏和卵巢组织中可检测到外源青鱼GH基因的转录.本研究成功获得了具有明显生长优势的P0代转青鱼GH基因异源四倍体鲫鲤,为建立转青鱼GH基因异源四倍体鲫鲤纯系和研制不育的转基因三倍体鱼奠定了基础.     

食物水平和初始体重对杂交罗非鱼生长和个体生长分化的影响

将72尾杂交罗非鱼分别养在12个水槽内,每个水槽内养6尾大小不同的鱼(A、B、C、D、E、F),其中鱼A的初始体重为62．69±1．46g,B为56．48±1．30g,C为50．75±1．19g,D为35．56±1．18g,E为31．05±0．88g,F为27。35±0．95g(平均值±标准误)．在4周实验中,实验鱼分别被停食或每天按体重1．5％、3．0％和饱食水平投喂．鱼的特定生长率(SGR)和食物效率(FE)先随食物水平增加而增加,当食物水平超过鱼体重的3．0％后,继续增加投喂量,SGR不再升高而FE明显下降．按体重1．5％投喂的鱼SGR和终体重个体间变异较大．对鱼A而言,食物水平超过体重1．5％后对其SGR无显著影响,但对鱼F而言,食物水平对SGR影响较大．结果表明,杂交罗非鱼的生长和个体生长分化与食物条件和初始体重有关,当食物水平超过体重的3．0％后,鱼的SGR较高,个体生长分化相对较轻．     

每期10题

1.人的胚胎以胎盘和脐带相连接,这样就使下面的哪种结构失去了重要性( )。 A.卵巢 B.卵黄 C.胎膜 D.子宫内膜 2.下面哪些物质直接影响女性月经周期子宫内膜的变化( )。 A.垂体分泌的调节性腺的激素 B.垂体分泌的生长激素 C.甲状腺分泌的激素 D 雌性激素和黄体素 3.男性精液中含有( )。 A.精子细胞和粘液 B.精子和粘液 C.精子和雄性激素 D.精子细胞和雄性激素 4.下面哪一项属于女性的第二性征( )。     

池塘混养模式下生态基对鲤生长及水质的影响

为探索池塘混养模式下生态基对鱼类生长的影响, 在6个土池中进行了一个饲养试验, 将土池分为2组, 一组为不放生态基的对照组, 另一组为放置生态基的生态基组, 每组3个重复。将尾均重为(310±11) g鲤3867尾、尾均重(810±15) g鲢及鳙370尾平均分别分为2组, 平均放养于6个土池中。对池塘鲤每天饲喂颗粒饲料3次, 饲养周期62d。在饲养期内, 每隔10天左右采集水样与底泥样品, 检测其中的浮游生物与微生物群落。在饲养结束后, 将试验鱼捕出并称重, 计算鲤的饲料效率。结果表明, 生态基组鲤鱼的增重率与饲料效率显著高于对照组; 生态基组的鲢、鳙末重显著低于对照组; 生态基组水体透明度与微生物群落多样性显著高于对照组; 生态基组浮游生物浓度低于对照组。结果表明, 在混养模式下土池中生态基的应用有利用促进鲤的生长, 然而池塘生态基的应用对于滤食性鲢、鳙的生长并无促进作用。     

Effects of oxytocin on semen release response in African catfish (Clarias gariepinus)

In silurid fishes, semen collection is practically impossible, even after hormonal stimulation. Instead, males are killed and testes macerated to obtain sperm. To understand the endocrine control of semen release in catfishes, we investigated the role of smooth muscle contractors in semen release and semen quality of African catfish (Clarias gariepinus). In in vitro experiments, testis slices were incubated with oxytocin (1 and 10 IU), isotocin (2 and 20 ug), vasopressin (0.2 and 2 ug), epinephrine (1 and 10 ug), PGF2alpha (1 and 10 ug), purified Clarias LH (300 ng) and partly purified Clarias pituitary extract (containing 300 ng LH). Only oxytocin increased sperm concentration of the medium (assessed by optical density measurements) compared to control incubations. Oxytocin was then tested in vivo in two groups of fish: normal males, and males that had been treated with 17alpha-methyltestosterone during larval stages to inhibit seminal vesicle development (MT males). Both groups of fish received two doses of carp pituitary suspension (8 and 10 mg/kg, respectively i.m.) with or without subsequent oxytocin treatment (5 IU/kg i.v.; cPS-OT treatment and cPS treatment, respectively). There was no effect of oxytocin on the number of strippable males. Of cPS and cPS-OT treated fish, 87% MT males and 60% normal males were strippable. The stripped semen volume was low in both groups but MT males produced higher (P < 0.001) hatching rates (63.1%) than did normal males (2.1%).     

鲤鱼不同品种（系）红血球抗原特异性的初步研究

用血球凝集和血清吸收试验的方法,对若干个鲤鱼品种及鲤鱼人工雌核发育系的红血球中的某些同种异型抗原纯合性进行了初步研究。①以抗雌核发育红鲤抗血清(abs／R)为试剂,三个雌核发育系(R8305,R8413,RMGF1)的RBC反应值分别为1521—2360,1521-2028,853—1521,普通红鲤、镜鲤、白鲢的反应值在80—782。②abs／8305,与abs／8413经靶细胞相互吸收后的残留活性显著降低(均为320),而以白鲢RBC吸收后抗血清活性不变。作者认为这部分抗原在鲤鱼种以下的品种和品系中具有特异性,可考虑将其作为鱼类品系鉴定的一种手段。     

鲤和草鱼杂交四倍体及其回交三倍体草鱼杂种的研究

进行了兴国红鲤♀×草鱼♂的杂交,杂种一代染色体数为142—156,众数值为146—152,系染色体自动加倍的异源四倍体。其外形和习性接近母本,两性均能成熟,雌、雄鱼分别与草鱼进行了回交。以草鱼为母本,杂种一代为父本的回交子代全部为草鱼形,草食性。以杂种一代为母本,草鱼为父本的回交子代一部分象草鱼,一部分象鲤,但草鱼形子代成活率较低,两个回交组合的子代均为三倍体,染色体数98—100,众数值98。1983年获得的杂种一代♀×草鱼♂的鲤鱼形回交杂种,已于1986年性成熟,雌、雄鱼分别与草鱼再次回交,获得了二次回交杂种。测定了红鲤、草鱼、杂种一代及其与草鱼回交子代的乳酸脱氢酶和过氧化物酶的同工酶,它们各有不同的电泳图谱。     

Sediment Raking as a Tool for Enhancement of Phosphate and Productivity of Water in Tropical Pond System

Changes of orthophosphate in water, induced by raking of surface sediment in various ways, were compared. Twelve limnocorrals were installed in a shallow mesotrophic pond using four treatments with triplicate replication:(1) raking of surface sediment (R) by means of a rake for 5min per week (2) fish (F), by introducing six common carp and two silver carp per limnocorral, (3) raking plus fish (R+F) and (4) control (C), without raking and fishless. After day 122, treatments were reversed in limnocorrals byremoving (netting) or introducing fish and stopping or initiating raking. Samples of water and sediment were collected from each limnocorral at weekly interval and monitored for orthophosphate (OP), primary productivity, phosphate solubilizing bacteria (PSB) and sediment available phosphorus. Raking in presence of fish caused 280 and 160% increase in OP concentrations in surface and bottom water, respectively, as compared with raking in absence of fish. Net effects of fish and raking were 13-81 and 26-241%, respectively in surface water and 48-100 and 46-95% in bottom water. This implied that in bottom water, net raking effect was 13-161% greater than net fish effect. Increase in PSB population, due to raking plusfish (R+F) was 6-82% higher than increase by fish alone (F). Responses of available-P in sediment were opposite to that of water, being highest in concentration in control (C) followed by fish (F), raking (R) and raking plus fish (R+F). The primary productivity showed variations parallel with that of orthophosphate of water. This suggests that raking accelerated the transformations of available-P fromsediment to overlying water mediated through oxygen-dependent sediment water exchange mechanisms.     

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10⁵ spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p < 0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p < 0.05).     

饲料蛋白水平对方正银鲫幼鱼生长、体成分、肝脏生化指标和肠道消化酶活性的影响

为研究饲料中不同蛋白水平对方正银鲫(Fang zheng Caucian carp)幼鱼生长、体成分、肝脏生化指标和肠道消化酶活性的影响, 配制蛋白水平为30.18%、33.13%、36.16%、39.34%、42.32%和45.46%的6种等脂等能饲料。选取初始质量为(3.10±0.01) g的方正银鲫幼鱼540尾, 随机分成6组, 每组3个重复, 每个重复30尾, 进行为期8周的饲养试验。结果表明: 随着饲料蛋白水平升高, 方正银鲫幼鱼增重率、特定生长率、饲料效率、蛋白质效率、全鱼粗蛋白含量、肝脏谷丙转氨酶和肠道胰蛋白酶活性均先升高后降低, 且在蛋白水平为36.16%时最大, 显著高于其他组(P<0.05); 肝脏总胆固醇和甘油三酯含量均呈逐渐下降趋势, 蛋白水平为30.18%的组显著高于除33.13%组以外的其他各组(P<0.05)。方正银鲫幼鱼特定生长率、蛋白质效率与不同蛋白水平分别进行二次回归分析得, 在试验条件下, 方正银鲫幼鱼饲料中最佳蛋白质水平为35.29%—37.07%。     

Seasonal short-term effects of naltrexone on LH secretion in male carp (Cyprinus carpio L.)

In order to evaluate the influence of the season (the stage of gonad maturity) on the modulatory role of endogenous opioid peptides in LH secretion in fish, sexually mature male carp (Cyprinus carpio L.) were intravenously injected with naltrexone-opioid receptor antagonist (5 or 50 microg kg(-1)) in the period of natural spawning (June) or gonad recrudescence (December). Moreover, the possible involvement of the dopaminergic system was studied in fish pre-treated with pimozide (dopamine receptor antagonist) and in intact fish. Blood samples were taken every minute, up to 10 min after naltrexone injection. In June, naltrexone significantly lowered LH levels in comparison to saline injected males. In December, there were no differences between saline and naltrexone-injected carps. In fish pre-treated with pimozide, neither in June nor in December were any significant differences in LH levels between control group and the groups injected with naltrexone found. The results showed that, in male carp, LH secretion under the influence of naltrexone depends on the stage of gonad maturity what suggests that the feedback of gonadal steroids on LH release could be mediated by the endogenous opioids. The role of dopamine in these processes is also discussed.     

用直接注射法生产转基因鱼

本文报道了对鲤鱼、鲫鱼受精卵不加任何去膜处理,用显微操作器把外源基因直接注射到卵核附近,构建转基因鱼的方法。本法操作方便,孵化条件简单,成活率高。斑点杂交和Southern Blot杂交结果表明,外源基因的整合率与其它方法构建的转基因鱼的外源基因的整合率相近。从1988年至今,本组运用这个方法生产转基因鲤鱼、鲫鱼一万余尾。     

红鲫(♀)×湘江野鲤(■)杂交一代生殖腺的细胞学研究

研究了以红色鲫鱼作母本和湘江野鲤作父本所得杂交一代(F_1)生殖细胞的生长发育。实验证明:(1)F_1雌性个体的生殖腺存在两种情况,一是完全能育型,占总数的44.3%;另一是发育不正常的类型,属两性嵌合体,占总数的55.7%。外观为雌性个体的两性嵌合体卵巢,既有卵细胞,也有精细胞。(2)F_1雄性个体的生殖腺出现三种不同的状况,一是完全能育型,只占总数的4.67%,可以完成由精原细胞到精子形成的全部发育过程;二是不完全能育型,占总数的61.7%,有少数精子细胞可以通过变态形成精子;三是完全不育型,这种类型包括有两性嵌合体(20.63%)和非两性嵌合体(13%),都不能产生成熟的精子。根据我们的实验结果证实,鲫(♀)、鲤((?))杂交所得的杂交一代(F_1)雄性个体,有部分是能育的,并已成功地获得了第二代(F_2)和第三代(F_3)。