桓仁林蛙与东北林蛙蝌蚪的形态特征及比较

桓仁林蛙（Rana huanrenensis Liu,Zhang,＆ Liu,1993）为一种与东北林蛙（Rana dybowskii）同域分布的林蛙,但其分布区域较狭窄.通过对其蝌蚪的形态特征描述及测量相关的形态学量度并和东北林蛙比较,发现在体色、体型、尾部形态以及身体各部位量度比值等方面存在显著性差异.桓仁林蛙的蝌蚪外形与流水型的蝌蚪相类似,而东北林蛙的蝌蚪则属于典型的静水型蝌蚪.     

Isolation and characterization of microsatellite markers from the Hypoptychus dybowskii (Pisces,Gasterosteiformes)

Hypoptychus dybowskii (Gasterosteiformes) exhibits allopaternal care frequently caused by various types of male reproductive tactics (sneaking, egg desertion and taking over). In order to understand this interesting reproductive system, we isolated microsatellites loci from H. dybowskii. Five microsatellites showed 2–10 alleles and expected heterozygosities ranged from 0.15 to 0.84. These were not significant deviations from Hardy–Weinberg expectations. These results suggest that these novel polymorphic loci should be useful for parentage analysis of H. dybowskii.     

东北林蛙皮肤中一类新抗菌肽cDNA的克隆及成熟肽的预测

为进一步研究和开发高效广谱天然抗生素的抗菌肽,本文克隆东北林蛙(Rana dybowskii)的抗菌肽基因,并预测其成熟肽的有关性质。根据蛙属抗菌肽信号肽末端序列设计简并引物,以RT-PCR技术扩增皮肤中抗菌肽的cDNA,并进行克隆测序。用生物信息学软件分析cDNA序列特点,预测成熟肽的理化性质。研究发现一种长度为28个氨基酸残基的新抗菌肽dybowsin-1,该肽具有Rana box结构;与已发现的抗菌肽仅有35%的同源性;理论等电点在9.70-10.01之间;均呈阳离子性;从第3或4个氨基酸开始到第16个氨基酸形成α-螺旋结构,极性氨基酸位于螺旋轮的一侧,非极性氨基酸位于螺旋轮的另一侧;具有N-端疏水、C-端亲水的两亲性。一个个体表达5条cDNA序列编码3种不同的dybowsin-1分子,显示出该抗菌肽表达的多样性。序列分析显示,该抗菌肽可能由多基因座位编码。     

中国林蛙Onconase样核糖核酸酶的基因克隆和表达

Onconase是从美洲豹蛙卵中提取的一种核糖核酸酶,由于其抗肿瘤活性而具有潜在的临床应用价值.以中国林蛙基因组为模板,克隆了一个新的RNase基因,并由此推导出了成熟林蛙RNase的氨基酸顺序.该酶是由103个氨基酸残基组成的,它保留了RNaseA家族成员酶催化活性必须的组氨酸和赖氨酸残基,以及CKXXNTF的序列特征,与Onconase具有73％的氨基酸顺序的相似性.林蛙酶比Onconue少一个氨基酸,成为选今为止发现的RNaseA家族中的最小成员;并且,林蛙酶拥有的精氨酸和酪氨酸残基比Onconase多3个.此外,在利用原核表达系统对林蛙RNase基因进行表达的过程中,表达产物对宿主显示出一定的细胞毒性.     

增温对人工养殖东北林蛙生长曲线的影响

2009年5～9月对东北林蛙增温和正常条件下的生长情况进行了初步研究.将一龄和二龄林蛙随机分成两组,一组在大棚内增温条件下饲养,另一组在围栏中常温条件下饲养,大棚内的平均温度较围栏中提升了3.17℃.2009年9月在大棚中饲养一龄林蛙和二龄雌性林蛙体重分别达到(6.30±2.62)g和(36.55±11.79)g,较围...     

桓仁林蛙和桓仁产东北林蛙的染色体组型

用骨髓细胞制片法对桓仁林蛙（Rana huanrenesis）和桓仁产东北林蛙（R．dybowskii）的染色体组型进行了报道,两种林蛙的染色体数均为2n=24,都可配成12对,按照相对长度可分为3组,A组包括第1～5对,为大型染色体（相对长度〉9．0）;B组是第6对,为中型染色体（相对长度在7．0～9．0之间）;C组包括第7～12对,这一组为小型染色体（相对长度〈7．0）。在两种林蛙的染色体组型中未发现有异型性染色体。桓仁林蛙的第1、3、4、5对为中部着丝点染色体,第9对为端部着丝点染色体,其余各对为亚中部着丝点染色体。东北林蛙的第1、2、3、4、5、6、8对为中部着丝点染色体,第9对为端部着丝点染色体,其余各对为业中部着丝点染色体。两种林蛙的第11对染色体长臂有明显的次缢痕。     

东北产三种林蛙血细胞的比较研究

应用Wright染色法对桓仁林蛙、东北林蛙和黑龙江林蛙的血细胞进行比较,将所得数据用SPSS软件统计。结果表明：桓仁林蛙、东北林蛙、黑龙江林蛙的血细胞形态、数量上存在差别,这可能与其生活环境和分类地位密切相关。     

Effects of anthropogenic intermixing on the genetic structure of Dybowski's frog populations in northeast China

The Dybowski's frog (Rana dybowskii) is mainly distributed in northeast China and the Russian Far East. Its dried oviduct, or frog oil, has been used in traditional Chinese medicine for thousands of years. Frogs from different geographic regions exhibit variations in their body mass and oil productivity. As a result, farmers usually import high quality breeding stock from different areas to increase oviduct productivity. This practice could alter the genetic structure of the frog populations. We used 10 microsatellite loci to investigate the level of genetic variation for 10 frog populations in the context of human-mediated disturbance. The study populations comprised the following: the Hebei, Tieli, and Jiamusi populations from the Lesser Khingan Mountains; the Mudanjiang, Jilin, Jiaohe, and Huadian populations from the north central Changbai Mountains; and the Tonghua, Benxi, and Xiuyan populations from the southern Changbai Mountains. Our results showed that the 3 Lesser Khingan Mountain populations exhibited highly significant differentiation values (F_ST = 0.047–0.071, P < 0.001). In addition, the Tonghua population differentiated significantly from the other 6 populations of the Changbai Mountains (P < 0.001). The genetic differentiation coefficient (F_ST) and gene flow (N_em) estimates showed that the Benxi and Jiaohe populations had a high degree of genetic similarity despite being geographically separated by mountains and river systems. We obtained similar results for the Xiuyan and Tieli populations. Furthermore, the degree of gene flow between the Jiamusi and the north central Changbai Mountain populations was greater than that observed between the Jiamusi and the other Lesser Khingan Mountain populations. These results therefore suggest that the Benxi, Xiuyan, and Jiamusi populations have undergone substantial intermixing because of human-mediated relocation. Economic demands make completely stopping the geographical relocation of Dybowski's frog in northeast China virtually impossible. Consequently, effective management strategies are now a high priority to ensure that conservationists and local economies can work together to guarantee the long-term survival of this species. © 2013 The Wildlife Society.     

中国林蛙核糖核酸酶Rdrlec新基因的原核可溶表达

来源于蛙属的核糖核酸酶由于具有显著的抗肿瘤活性而备受关注,Rdrlec是从中国林蛙基因组中克隆得到的核糖核酸酶新基因。获得大量高纯度野生型重组蛋白是研究其功能的基础。按照大肠杆菌偏好的密码子人工合成Rdrlec基因,通过EcoR I和Hind III位点插入到表达载体pET-32a(+)中构建pET32-Rdrlec重组表达质粒,转化到Escherichia coli BL21(DE3)中,0.4 mmol/L IPTG 30℃诱导6 h后,融合蛋白主要以可溶形式表达,经过Ni-NTA亲和纯化和Sephadex G75层析纯化,得到电泳纯融合蛋白。肠激酶切割后得到Rdrlec野生型重组蛋白,具有降解RNA的酶活性,证明分子的空间结构已经正确形成。Rdrlec野生型重组蛋白表达成功,为后续蛋白结构与功能的研究以及进一步的开发应用提供了原料。