Construction of a cDNA Library from the Testis and Sequence Analysis of the Ubiquitin Gene from Rana nigromaculata

A full-length cDNA library from the testis of dark-spotted frogs （ Rana nigromaculata ） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the testis and reverse transcripted into full-length cDNA using PowerScript reverse transcriptase. The first-strand cDNA was amplified using long-distance PCR （LD-PCR）. After Sfi Ⅰ digestion and fractionation, cDNA （ 〉 500 bp） was ligated to λ TriplEx2 vector and packaged with GigapackⅢ Gold Packaging Extract. The titers of optimal primary libraries were 2.0×10^6 pfu/mL and 2.4 × 10^6 pfu/mL and the tlters of the amplified libraries were 0.48 × 10^9 pfu/mL and 3.0 × 10^9 pfu/ mL, respectively. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. The libraries were converted into pTriplEx2 plasmids in E. coli BM 25.8 strain. The insert sizes were measured by PCR which showed most fragments were over 500 bp and the average length was 1.0 kb approximately. A positive clone of 1 171 bp was sequenced and named RnUb based on sequence similarity with the known ubiquitin genes in Gen- Bank. This sequence was a full-length cDNA with complete coding sequences, which indicated that the library built a base for screening the full-length cDNA. These data showed that this library attained to the requirements of a standard cDNA library. This library provided a useful resource for the functional genomic research of Rana nigromaculata.

黑斑蛙(Rana nigromaculata)不同组织器官和不同发育时期同工酶的比较研究

采用聚丙烯酰胺凝胶平板电泳 (PAGE) ,对黑斑蛙的五种不同组织器官和五种不同发育时期的个体进行了酯酶 (α—EST)和过氧化物酶 (PO)同工酶的差异比较研究 .结果显示 ,不同组织器官中两种同工酶均存在明显的区别 ;不同发育期的α—EST同工酶也存在质的差异 .从蝌蚪到幼蛙的发育中 ,α—EST同工酶谱逐渐增加 ,但从幼蛙发育到成体蛙 ,α—EST同工酶谱却出现了减少现象 .不同发育时期的个体Rf为 0 .2 3的α—EST酶谱皆得以表达 .

Adaptive changes of gastro-entero-pancreatic system endocrine cells in the black-spotted pond frog Rana nigromaculata after fasting

Changes in distribution density,morphology and secretory content of endocrine cells in the gastro-entero-pancreatic system of black-spotted frogs Rana nigromaculata before and after fasting were investigated using immunohistochemistry and antisera to six gut hormones.Six types of endocrine cells were detected in the digestive tract of Rana nigromaculata,including 5-hydroxytryptamine(5-HT),gastrin(GAS),somatostatin(SOM),glucagon(GLU),pancreatic polypeptide(PP)and vasoactive intestinal polypeptide(VIP)cells.After fasting,the density of 5-HT cells in the esophagus,cardia and fundus,GAS cells in the fundus and pylorus,PP cells in the fundus decreased significantly(P<0.01),while SOM cells in the cardia,GLU cells in the rectum increased significantly(P<0.01).The cytoplasmic processes of 5-HT cells became shorter or not detectable.The secretory content of GAS cells reduced in the cardia.The positive immunostaining reaction in the perinuclear region of SOM cells in the cardia,fundus and pylorus became weaker,while the staining intensity in the periphery of these cells became stronger.VIP cells were not detectable in the whole digestive tract after fasting.Five types of endocrine cells were found in the pancreas of Rana nigromaculata,including 5-HT,GAS,SOM,GLU and PP cells.After fasting,the density of 5-HT cells decreased slightly(P>0.05),while SOM,GAS,GLU and PP cells increased significantly(P<0.01).Furthermore,the secretory content of GLU cells increased significantly.Considering their functionalities,our results indicate that the changes of GEP endocrine cells in Rana nigromaculata responded adaptively to starvation-induced stress.

黑斑蛙(Rana nigromaculata)早期发育过程中乳酸脱氢酶同工酶

用垂直平板聚丙烯酸胺凝胶电泳技术对黑斑蛙早期胚胎发育过程中乳酸脱氢酶（LDH）同工酶进行研究。结果表明，黑斑蛙胚胎各个不同发育时期LDH同工酶谱有很大差异：LDH1在整个胚胎发育过程中均处于活化状态，LDH2、LDH4在原肠中期开始出现，LDH3在尾芽期开始出现。从尾芽期到尾血循环期只出现LDH－1、－2、－3和-4四条酶带，但不同时期各种同工酶的表达活性不同。

Skeletochronological Study of Age,Longevity and Growth in a Population of Rana nigromaculata(Amphibia:Anura) in Sichuan,China

A skeletochronological study was conducted for the age, longevity and growth of a Rana nigromaculata population in northeastern Sichuan, China. Of 139 specimens, 119(68 males and 51 females) exhibited distinct arrested growth lines in phalanges. Age at first reproduction was found to be two years of age for males and three for females. Maximum longevity was estimated to be at least six years in males and seven years in females. Average age did not differ between males and females. Breeding females were significantly older than breeding males. Females were larger in body size and heavier in body mass than males. Age was not correlated with body size and body mass in males, but a significant correlation was found among age, body size and body mass in females. The growth curve appeared significantly different between the sexes, with a higher growth rate throughout life and a larger asymptotic size in females. Moreover, analysis of the growth model indicated that the population studied was relatively stable.

New Efficient Ecological Breeding Technology of Rana nigromaculata

High density cell intensive culture pattern is an ecological, efficient, planting and breeding combined new breeding pattern of Rana nigromaculata. It protects racial continuation and population expansion of R. nigromaculata, beneficial for environment construction. Meanwhile, it greatly increases the farmers＇ income, conducive to economic development. The economic, ecological and social benefit of this culture pattern is remarkable.

黑斑蛙Rana nigromaculata正常发育表

在20℃下黑斑蛙由受精卵发育到鳃盖合缝封闭时止共需202.3±5.9小时。为了记载和研究的方便我们根据外部形态和生理的现象有时根据时间将这段发育的期间分为26个时期。

黑斑蛙(Rana nigromaculata Hallowell)肾上腺的组织化学观察

关于蛙肾上腺的组织学结构已有不少作者[1,2,3]作了详细的观察,一致认为它由(1)肾上腺细胞;(2)肾间腺细胞;(3)Stilling氏细胞;(4)神经节细胞等四种细胞混合构成。无皮质及髓质之分。并指出肾上腺细胞和肾间腺细胞分别与哺乳动物肾上腺的髓质细胞和皮质细胞相符合;而Stilling氏细胞在哺乳类肾上腺中没有发现相应的成分。

Molecular characterization and m RNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Like Xenopus laeuis, some species of the Rang genus are also used to study endocrine disrupting chemicals （EDCs）. Although ribosomal protein L8 （rp18） is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rang, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rp18 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rp18 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rp18 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rp18 gene was found in all tissues examined （brain, liver, intestine, tail, testis and ovary） during R. nigromaculata development. Finally, we investigated rp18 expression under exposure to hormones. No change in rp18 mRNA expression was found under exposure to thyroid hormone （T4） and estrogen （estradiol）, whereas expression of the corresponding biomarker genes was induced. Our results show that rp18 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rp18 as a reference gene in other Rang species due to the high conservation of rp18 among the Rana genus.

Molecular characterization and developmental expression patterns of thyroid hormone receptors(TRs) and their responsiveness to TR agonist and antagonist in Rana nigromaculata

Considering some advantages of Rana nigromaculata as an experimental species, we propose that this species, like Xenopus laevis, could be used to assay thyroid hormone（TH） signaling disrupting actions. To validate the utilizability of R. nigromaculata, we investigated the responsiveness of R. nigromaculata to a TH receptor（TR） agonist（T3） and antagonist（amiodarone） by analyzing expression, based on characterizing TR cDNA and developmental expression patterns. With high levels of identity with the corresponding genes in X. laevis, both TRα and TRβ in R. nigromaculata exhibited roughly similar developmental expression patterns to those of X. laevis, in spite of some species-specific differences. Both TRα and TRβ expression had greater changes in the liver and intestine than in the tail and brain during metamorphosis. T3 exposure for 2 days induced more dramatic increases of TRβ expression in stage 27 than in stage34 tadpoles but not in stage 42 tadpoles, showing that the responsiveness of R. nigromaculata to TH decreased with development and disappeared at the onset of metamorphic climax.Corresponding to greater changes of TRβ expression in the liver and intestine than in the tail and brain during metamorphosis, the liver and intestine had higher responsiveness to exogenous T3 than the tail and brain. Amiodarone inhibited T3-induced TRβ expression. Our results show that R. nigromaculata can be used as a model species for assaying TH signaling disrupting actions by analyzing TRβ expression, and intestine tissues at stage 27 are ideal test materials due to high responsiveness and easy accessibility.

Breeding ecology and oviposition site selection of blackspotted pond frogs (Rana nigromaculata) in Ningbo, China

The black-spotted pond frog(Rana nigromaculata)is one of the most widely distributed species in China.However,there have been only a few qualitative descriptions of their breeding ecology and oviposition site selection.From 2004 to 2006,we investigated the breeding ecology and oviposition site selection of R.nigromaculata in Ningbo,China,quantitatively.Analyses of breeding ecology show that:(1)mean frog density in the breeding season was 0.0903±0.0029 individuals per meter(n=11)(mean±SE);(2)R.nigromaculata was a sexually dimorphic species,with females significantly larger than males in both body weight and snout-vent length;(3)the clutch size averaged 4643.04±235.96 eggs(range 1546–7897,n550);and(4)the egg size ranged from 1.50 to 1.74 mm in diameter,with an average egg size of 1.6050±0.0046 mm(n=226).Oviposition sites differed significantly from random sites in percentages of water,bare ground and vegetation cover,water depth(cm),water temperature(uC)and water turbidity.Rana nigromaculata preferred microhabitats with higher percentages of water and vegetation cover,while it avoided microhabitats with deeper water.The results suggest that microhabitats with higher percentages of water and vegetation cover,but not deeper water,should be priorities for protection and conservation of the breeding habitats of R.nigromaculata.

冻结方式对黑斑蛙后腿肌肉持水性及肌原纤维蛋白结构特性的影响

本研究采用-20℃冰柜冻结、-50℃冰柜冻结、-50℃液氮冻结、-80℃液氮冻结处理黑斑蛙后腿,探究不同冻结方式及冻结温度对黑斑蛙后腿肌肉持水性及肌原纤维蛋白结构的影响。结果表明:-20℃冰柜冻结、-50℃冰柜冻结、-50℃液氮冻结、-80℃液氮冻结组通过最大冰晶生成带(-1~-5℃)的时间分别为91.5、57.0、1.5、0.5 min。相比两组冰柜冻结组,两组液氮冻结组的解冻损失率降低,不易流动水含量升高,自由水含量下降;相比新鲜样品,冷冻后黑斑蛙后腿的肌原纤维持水性、蛋白溶解度、总巯基含量、内源荧光强度显著下降,蛋白粒径、表面疏水性、无规卷曲相对含量呈现升高趋势。其中-50℃液氮冻结组的肌原纤维持水性、蛋白溶解度、总巯基含量、内源荧光强度分别下降了8.10%、9.57%、10.50%、67.36%,体积平均粒径、表面疏水性、无规卷曲相对含量分别增加了100.05%、82.24%、13.65%,变化幅度显著低于其他实验组(P<0.05)。-80℃液氮冻结组黑斑蛙后腿肌原纤维蛋白变性程度显著低于-20℃冰柜冻结组,但高于-50℃液氮冻结组(P<0.05)。扫描电子显微镜观察结果显示,-50℃冰柜冻结和-50℃液氮冻结组肌纤维排列较为紧密规则,-80℃液氮冻结的黑斑蛙肌纤维排列规整但有轻微裂隙,-20℃冰柜冻结组肌纤维出现明显破裂。综上,液氮冻结可减缓黑斑蛙后腿肌肉冷冻过程中水分迁移,有效抑制黑斑蛙肌原纤维蛋白的冷冻变性,其中-50℃液氮冻结效果优于-80℃液氮冻结。

黑斑蛙不同放养密度对稻蛙共作水稻生长性能的影响

开展了黑斑蛙不同放养密度对稻蛙共作水稻生长性能的影响试验。分别于水稻的返青期、分蘖期、抽穗期、成熟期,测量水稻株高、分蘖数、叶绿素含量和净光合速率等指标。设置水稻单作(RM)、稻蛙共作(RF)2种模式。黑斑蛙蝌蚪设置11个处理组,分别为RF5、RF6、RF7、RF8、RF9、RF10、RF11、RF12、RF13、RF14和RF16,对应投放密度为每667 m25万、6万、7万、8万、9万、10万、11万、12万、13万、14万和16万尾。结果表明,稻蛙共作模式的水稻株高、分蘖数、叶绿素相对含量以及净光合作用速率,均显著高于水稻单作模式,黑斑蛙蝌蚪投放密度为每667 m28万尾处理组的水稻株高、分蘖数、叶绿素相对含量以及净光合速率,显著高于其他处理组。指出,黑斑蛙蝌蚪每667 m28万尾为最佳投放密度,可以作为贵州山区稻蛙共作生态种养模式推广应用的参考投放密度。

不同黑斑蛙蝌蚪放养密度对贵州山区稻蛙共生模式中黑斑蛙生长的影响

本研究旨在探讨不同放养黑斑蛙蝌蚪对稻蛙共生模式中黑斑蛙生长的影响,本研究采用田间试验方法,分别设置75只/m^(2)、105只/m^(2)、135只/m^(2)、165只/m^(2)和195只/m^(2)五种蝌蚪放养密度,比较了不同蝌蚪放养密度下黑斑蛙个体的生长差异,建立了体长、体质量与养殖时长的生长模型,进一步拟合了体长、头宽、后肢长与体质量的关系。结果表明:不同蝌蚪放养密度对体长、头宽、后肢长和体质量均有显著影响(P<0.05);体长、头宽、后肢长均与体质量存在较高的相关性(P<0.05),拟合度达0.956 3;75只/m^(2)和135只/m^(2)组黑斑蛙的体质量与养殖时长间的生长曲线(?)值均为2.33,显著高于165只/m^(2)和195只/m^(2),且体质量分布中,40g以上的黑斑蛙个体重比例最高的为135只/m^(2),高于其他处理组。从生长角度考虑稻蛙共生模式中黑斑蛙合理的放养密度为在135只/m^(2)左右。

中西药联用对黑斑蛙源“歪头病”病原菌的体外抑菌效果研究

为研究常用抗生素和中草药对黑斑蛙源“歪头病”病原菌米尔伊丽莎白菌和肺炎克雷伯菌的体外抑菌效果,采用纸片扩散法和二倍稀释法测定了米尔伊丽莎白菌和肺炎克雷伯菌对9种常用抗生素和6种中草药的敏感性,棋盘法测定两联药方的部分抑菌浓度(FIC)。结果显示,阿米卡星、多粘菌素B和环丙沙星对米尔伊丽莎白菌和肺炎克雷伯菌的抑菌效果较好,最小抑菌浓度(MIC)分别为2.5μg/mL、5-10μg/mL和10μg/mL;五倍子、金诃子和金银花抑菌效果较好,MIC分别为31.25 mg/mL、125 mg/mL和500 mg/mL;这三种抑菌效果较好的中草药之间两两联用未发生拮抗作用。中西两联药方中,五倍子和多粘菌素B对米尔伊丽莎白菌的抑菌效果较好,FIC为1,表现为相加作用,可作为黑斑蛙“歪头病”抑菌药物的参考配伍组合。

黑斑蛙歪头病的病原菌分离鉴定及其感染病理分析

本研究从具有典型歪头病症状的濒死黑斑蛙(Rana nigromaculata)肝脏中分离到一株优势菌HJS0526,以确定常德地区黑斑蛙歪头病的病原菌及分析其致病机理。通过细菌形态学、生理生化分析、16S rDNA序列比对确定HJS0526菌株为米尔伊丽莎白菌(Elizabethkingia miricola)。人工感染HJS0526菌株14 d后,实验组歪头症状出现率达90%以上,死亡率高于50%,感染症状和自然发病症状基本一致。药物敏感试验结果表明,菌株HJS0526对环丙沙星和氟苯尼考高度敏感,金银花和鱼腥草醇提取液对HJS0526菌株具有极好的抑制效果;组织病理学研究结果显示,HJS0526菌株感染造成黑斑蛙全身多组织器官功能障碍,特别是肝、脾、肾和肺等组织严重损伤,表现为明显充血、出血及细胞变性、坏死。米尔伊丽莎白菌对黑斑蛙具有高致病性,感染后可导致黑斑蛙多组织器官功能损伤而死亡。本研究结果可为预防和控制常德地区由米尔伊丽莎白菌引起的黑斑蛙歪头病提供科学依据。

黑斑蛙歪头病病原米尔伊丽莎白菌的分离、鉴定及特性分析

为查明湖北石首大垸农场养殖的黑斑蛙(Rana nigromaculata)歪头病病原与致病原理,本研究应用微生物分离技术,从病蛙脑、肝、脾和肾脏组织中分离、纯化病菌,采用形态特征、生理生化鉴定、16S rRNA基因序列分析进行细菌种类鉴定,通过组织病理学方法分析患病蛙病理损伤,及人工回归感染确定其致病性,并利用纸片扩散法进行药物敏感性试验。结果显示:分离菌(BSH3)与米尔伊丽莎白菌(Elizabethkingia miricola)聚为一支,结合该菌的形态学特征和生理生化特征,综合鉴定为米尔伊丽莎白菌。组织病理切片结果表明分离菌(BSH3)会导致蛙脑组织基质水肿,基质中出现空泡和炎性细胞浸润的现象;肝、脾和肾脏组织出现变性、坏死和炎症细胞浸润。人工回归感染试验显示,菌株可使黑斑蛙出现与自然发病蛙相似症状,半数致死浓度为6.3×10^(4) CFU/mL。药敏试验结果表明菌株已呈现多重耐药性,仅对少数检测抗生素(米诺环素、氟苯尼考、利福平和万古霉素)敏感,根据药敏试验结果及农业部水产用药规定,推荐使用氟苯尼考治疗该疾病。

黑眶蟾蜍和黑斑蛙消化道5-羟色胺免疫活性细胞的免疫组织化学

对黑眶蟾蜍(Bufomelanostictus)和黑斑蛙(Rananigromaculata)消化道5-羟色胺免疫活性细胞的形态、分布进行了免疫组织化学定位。5-羟色胺免疫活性细胞在黑眶蟾蜍和黑斑蛙消化道各段中均有分布,分布密度均呈升-降-升-降的波浪式分布特点,二者在幽门部和回肠都有个分布的高峰值。黑眶蟾蜍回肠最高,空肠、幽门部次之,十二指肠、直肠最低;黑斑蛙幽门部最高,回肠、空肠次之,食道、贲门部、直肠最低。5-羟色胺免疫活性细胞位于胃的胃腺上皮、食道及肠的粘膜上皮,有圆形、椭圆形、梭形、楔形等,有的有胞突。文中讨论了5-羟色胺免疫活性细胞分布型的原因及形态与功能的关系。

镉长期暴露对黑斑蛙的氧化胁迫和抗氧化能力的影响

在实验条件下,将黑斑蛙暴露于12.5 mg/L和25.0 mg/L浓度的镉溶液中30d,分别测定了黑斑蛙在暴露10、20和30d时肝、肾组织中镉(Cd)含量、过氧化产物丙二醛(MDA)的含量、还原型谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)活性,以探讨镉对机体的脂质过氧化作用及机体的抗氧化损伤机制。实验结果表明,不同剂量组黑斑蛙肝、肾镉含量、MDA含量均随着镉暴露时间的延长而升高,且肝MDA含量与镉在肝中的蓄积量呈显著正相关(R2=0.8643,n=9)。肝脏GSH含量随镉暴露时间的延长而被显著诱导,且与MDA含量呈显著正相关(R2=0.5933,n=9);肾GSH含量则随暴露时间的延长而显著下降,与MDA含量呈显著负相关(R2=0.8609,n=9)。不同剂量组肝SOD活性随镉暴露时间的延长而升高,肾SOD活性在高剂量组随镉暴露时间的延长表现为先升高后回落下降的趋势。可见,在镉的长期暴露下,细胞膜过氧化增强是镉伤害机体的主要原因,而GSH含量、SOD活性的升高则可能是机体抗过氧化的机理之一。

中国黑斑蛙种群的线粒体DNA多样性和生物地理演化过程的初探

本文通过对黑斑蛙 8个地方种群 2 5 7个标本的线粒体基因组Cytb基因片段的序列分析 ,探讨了其种群遗传多样性的现代分布格局及其生物地理演化过程。对所检测出的 79个单倍型的分析表明 :黑斑蛙在我国分布区内中部地区的种群遗传多样性高于南北边缘种群。种群间基因流同地理隔离距离呈显著负相关 ,种群遗传分化符合距离隔离或脚踏石模型。线粒体单倍型以广西和四川种群的个体的遗传分化为最深 ,反映出西部山区的屏障隔离效应。单倍型歧点分布分析表明 ,约 10 3- 2 2 6万年前 ,即在末次间冰期 ,黑斑蛙在其分布范围内经历过一次大规模的种群扩张事件。单倍型的分布格局和共享情况显示黑斑蛙种群在冰期后的重新扩张过程主要表现为由南往北的递进式扩散形式。第四纪冰期气候的反复波动导致了黑斑蛙适宜生境在纬度方向上的平移 ,进而导致黑斑蛙种群随生境变化而进行扩张或收缩 ,使黑斑蛙种群的遗传多样性能始终保持在分布区中间高而南北两端低 。