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...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.展开更多
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 imm...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.展开更多
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 arreste...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.展开更多
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. nigrom...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.展开更多
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 ...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.展开更多
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 t...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.展开更多
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 selectio...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.展开更多
基金The National Natural Science Foundation of China(30640048)the Natural Science Foundation in Anhui Province(01043202)
文摘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.
基金supported by Open Fund of Key Laboratory of Bio-Macromolecular Evolution(No.2006B001)Foundation for outstanding academic team of Zoological Biology in Anhui Normal UniversitySpecial Foundation for Provincial Key Laboratories of Biotic Environment and Ecological Safety and Key Laboratory for Conservation and Use of Important Biological Resources of Anhui Province
文摘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.
基金Financial support was provided by the Foundation of Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education)
文摘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.
基金Supported by Innovative Entrepreneurial Training Project of National College StudentsFund Project of Zhujiang College of South China Agricultural University(201512623001)
文摘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.
基金supported by the National High Technology Research and Development Program (863) of China (No. 2012AA06A302)the Public Welfare Research Project for Environmental Protection (No. 201109048)the National Natural Science Foundation of China (No. 21077125)
文摘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.
基金supported by the Public Welfare Research Project for Environmental Protection (No. 201109048)the National High Technology Research and Development Program (863) of China (No. 2012AA06A302)the National Natural Science Foundation of China (No. 21077125)
文摘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.
基金The project was supported by the National Natural Science Foundation of China(Grant No.30270246)the CAS Innovation Program(No.KSCX3-IOZ-02 and KSCX2-SW-118).
文摘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.
