青岛文昌鱼神经胚中期cDNA文库的构建

提取青岛文昌鱼18小时神经胚中期mRNA,以5＇脱磷的NotI-oligo(dT)18为引物,反转录合成cDNA.双链cDNA的5＇端钝端连接上带EcoRI突出末端的衔接头,再经NotI酶切,在cDNA的3＇端形成NotI突出末端.以SizeSep离心层析柱除去400bp以下的小分子,与带有NotI和EcoRI突出末端并经过5＇端脱磷的λExCellNotI/EcoRI/CIP载体DNA进行连接,经体外包装和感染NM522宿主菌,得到了3.6×10     

青岛文昌鱼NADH脱氢酶亚基I基因片段的克隆

NADH脱氢酶亚基I是细胞电子传递链的主要成员之一，采用简并引物PCR方法获得青岛文昌鱼NADH脱氢酶亚基I基因片段，将基氨基酸序列与其他生物如佛罗里达文昌鱼，斑马鱼，爪蟾等无脊椎和脊椎动物NADH脱氢酶亚基I基因相应片段进行了同源性分析，均显示较高的同源性，研究结果证实青岛文昌鱼作为脊索动物的代表之一，与脊椎动物有着较近的亲缘关系，是从无脊椎动物到脊椎动物的过渡类型。     

青岛文昌鱼肾上腺髓质素基因的克隆、组织特异性表达和生物信息学分析

用PCR技术在青岛文昌鱼中克隆到肾上腺髓质素(AM)基因的全长,命名为BjAM基因.序列分析发现BjAM基因全长1805bp,开放阅读框366bp,5'-UTR为82bp,3'-UTR为595bp;进行生物信息学分析发现,BjAM蛋白序列具有AM家族成员典型的保守关键位点,预测BjAM与AM1是直系同源,与脊椎动物AM1具有类似的生物学功能.组织特异性表达分析发现BjAM在各个组织中均有表达,鳃、脊索、肝盲囊和后肠中的表达量较高.头索动物文昌鱼中AM基因克隆和生物信息学分析为进一步构建原核表达载体、探究AM蛋白的功能奠定了基础.     

秦皇岛昌黎邻近海域青岛文昌鱼资源调查及大型底栖动物群落特征研究

为了解秦皇岛昌黎邻近海域国家二级保护动物青岛文昌鱼(Branchiostoma tsingdauense)的资源现状,科学评价该海域底栖环境生态健康状况,于2021年春、夏两季在昌黎邻近海域设置24个站位进行大型底栖动物及环境要素的调查监测,并使用香农-威纳(Shannon-Wiener)多样性指数及AZTI海洋生物指数(AMBI)、多变量AZTI海洋生物指数(M-AMBI)对昌黎邻近海域大型底栖动物群落健康状况进行评价。调查结果表明：2021年夏季昌黎邻近海域青岛文昌鱼平均生物密度为5 ind./m²,处于近年调查的最低水平。春、夏两季昌黎邻近海域青岛文昌鱼均以Ⅲ龄鱼为主,种群年龄结构出现异常现象。春、夏两季共采集到大型底栖动物89种,两季大型底栖动物群落结构存在一定差异,物种更替率较高。两季大型底栖动物平均生物密度为138 ind./m²,平均生物量为20.95 g/m²,平均Shannon-Wiener多样性指数为2.11。Shannon-Wiener多样性指数及AMBI、M-AMBI指数等生态健康评价方法评价结果...     

家蚕促咽侧体素受体基因克隆及发育表达

文章克隆得到家蚕(Bombyx mori)神经肽促咽侧体素受体基因(Allatotropin receptor,BommoATR),开放阅读框全长为1 254 bp,编码416个氨基酸.Bommo-ATR氨基酸序列的二级结构预测为7次穿膜蛋白,符合GPCR家族成员的典型特征.实时荧光定量PCR结果表明,家蚕脑-咽下神经节复合体中的ATR mRNA在5龄幼虫期表达量最高,其次为蛹后期,蛹前期和成虫阶段表达量最低.其中5龄幼虫第2天表达量最高,1～5 d持续维持较高的表达水平,这可能与保幼激素的合成有关.     

一株(鱼免)鱼溃疡致病菌的分离及鉴定

该研究从宁波市象山港(鱼免)鱼养殖区患病(鱼免)鱼肾脏中分离到1株优势菌Mm041,人工回感试验确定该菌株为致病菌株,测定其对(鱼免)鱼的半致死量为3.0×106 CFU.通过形态学观察、生理生化特征鉴定,并结合细菌16S rRNA基因序列同源性和系统发育分析,确定Mm041为美人鱼发光杆菌.药敏试验结果表明,该菌株对...     

TAT-GFP-ERβ融合蛋白的原核表达与鉴定

从前列腺中提取总RNA,RT-PCR获得ERβ基因,将其联人pEGFP载体,得到重组质粒pEGFP-ERβ,再将上述质粒的GFP-ERβ片段亚克隆于pTAT-2.1质粒,形成pTAT-GFP-ERβ重组质粒.在BL21工程菌中IPTG诱导表达TAT-GFP-ERβ融合蛋白.用Western Blot鉴定纯化的融合蛋白的免疫原性.用纯化的蛋白转导事先转染ERE-Luc报道质粒的Hela细胞,用Luciferase Assay鉴定转导蛋白的生物学活性.证明TAT-GFP-ERβ蛋白具有完整的生物活性.     

青岛文昌鱼硫氧还蛋白基因的克隆及同源性分析

通过对文昌鱼神经胚cDNA文库进行测序 ,获得含文昌鱼硫氧还蛋白基因全部读框的cDNA序列 ,并演绎出对应的氨基酸序列 ,对其可编码蛋白进行了分析预测 ,还与多种脊椎动物和无脊椎动物中同种蛋白的同源性进行比较分析 .发现该蛋白具有典型的硫氧还蛋白活性部位 ,与脊椎动物硫氧还蛋白的同源性大于无脊椎动物 ,说明作为脊椎动物和无脊椎动物之间典型的过渡类型 ,文昌鱼在进化上更接近于脊椎动物 .     

水稻脱落酸受体PYL2与mNeonGreen融合蛋白的原核表达及纯化

目的:为建立脱落酸的荧光检测方法.方法:通过重叠PCR技术,在水稻脱落酸受体PYL2蛋白的C-端融合一种新型黄绿色荧光蛋白mNeonGreen,并对融合蛋白进行原核表达和纯化.结果:获得了高纯度的融合蛋白PYL2-mNeonGreen,加入脱落酸后,该融合蛋白黄绿色荧光强度逐步降低,表明融合蛋白PYL2-mNeonGreen能特异性识别脱落酸.结论:该研究为后续脱落酸的原位检测奠定了基础.     

鼠源抗过敏融合蛋白的瞬时表达及纯化

采用流加和降温培养工艺,实现在5 L搅拌式生物反应器规模的瞬时基因表达。首先比较了两种表达载体在CHO-S细胞中的瞬时基因表达,发现pID-mAAFP载体介导的瞬时转染表达可以获得mAAFP的高水平表达。采用pID-mAAFP载体在1.3 L搅拌式生物反应器中进行瞬时基因表达,与分批培养相比,通过流加和降温培养工艺对转...     

Position in the evolution of reproductive endocrine of amphioxus,Branchiostoma belcheri

This review summarizes the recent discoveries of many authors who found that in amphioxus Hatschek’s pit is capable of synthesizing vertebrate gonadotropin-like substance, and that the content of gonadotropin-releasing hormone in the amphioxus’ body shows a positive correlation with the reproductive cycle, and that the sex steroid hormone exists in gonads. Exogenous hormones could promote gonadal development, maturation and reproductive activity in amphioxus. A possible implication might be that the reproductive activity in amphioxus is regulated by reproductive hormones like vertebrate, indicating the existence of primitive reproductive endocrine regulatory axis, brain vesicle-Hatschek’s pit-gonads axis, as compared with regulatory axis of vertebrate. It will provide a new line for establishing the position of reproductive endocrine evolution in lancelet.     

Distribution of CRH- and ACTH-like immunoreactive cells in amphioxus, Branchiostoma belcheri

Immunocytochemical studies on the nervous system, Hatschek’s pit，digestive tract and gonads tissues of an amphioxus (Branchiostoma belcheri) were performed using polyclonal antibodies against human corticotrophin-releasing hormone (CRH) and human adrenocorticotropin (ACTH). The results showed that many CRH-like immunoreactive neurons were distributed specifically on the dorsal side and ventral side of brain vesicle, while a few CRH-like neurons and their fibers in spinal cord. At the same time, the epithelial cells in the basic region of Hatschek’s pit were shown immunopositive to CRH antibody. In gonads (ovary and testis), CRH-immunopositive substance was localized in the cytoplasm near oocyte nucleus and in early spermatogenic cells. ACTH-like immunoreactivities were observed specially in the neurons and their protrusions localized on the ventral side of the brain vesicle and in spinal cord, and also in epithelial cells of Hatschek’s pit, enteric neurons of digestive tract, oocytes in ovary and in early spermatogenic cells as well. It was found for the first time that CRH-like neurons existed in the middle region of brain vesicle (corresponding to the hypothalamus of vertebrates) and ACTH-like immunopositive cells existed in Hatschek’s pit, implying that a control mechanism between brain vesicle and Hatschek’s pit maybe had been already built in amphioxus as that in vertebrates. The present study will provide new morphological evidence for the origin and evolution of ACTH. In addition, the immunoreactivities of CRH and ACTH in the digestive tract and gonads suggested other physiological function of CRH and ACTH in amphioxus.     

Distribution of CRH-and ACTH-like immunoreactive cells in amphioxus,Branchiostoma belcheri

Immunocytochemical studies on the nervous system,Hatschek's pit,digestive tract and gonads tissues of an amphioxus(Branchiostoma belcheri)were performed using polyclonal antibodies against human corticotrophin-releasing hormone(CRH)and human adrenocorticotropin(ACTH).The results showed that many CRH-like immunoreactive neurons were distributed specifically on the dorsal side and ventral side of brain vesicle,while a few CRH-like neurons and their fibers in spinal cord.At the same time,the epithelial cells in the basic region of Hatschek's pit were shown immunopositive to CRH antibody.In gonads(ovary and testis),CRH-immunopositive substance was localized in the cytoplasm near oocyte nucleus and in early spermatogenic cells.ACTH-like immunoreactivities were observed specially in the neurons and their protrusions localized on the ventral side of the brain vesicle and in spinal cord,and also in epithelial cells of Hatschek's pit,enteric neurons of digestive tract,oocytes in ovary and in early spermatogenic cells as well.It was found for the first time that CRH-like neurons existed in the middle region of brain vesicle(corresponding to the hypothalamus of vertebrates)and ACTH-like immunopositive cells existed in Hatschek's pit,implying that a control mechanism between brain vesicle and Hatschek's pit maybe had been already built in amphioxus as that in vertebrates.The present study will provide new morphological evidence for the origin and evolution of ACTH.In addition,the immunoreactivities of CRH and ACTH in the digestive tract and gonads suggested other physiological function of CRH and ACTH in amphioxus.     

Synergistic actions of complement and lysozyme in clearance of Escherichia coli from amphioxus Branchiostoma belcheri

Amphioxus appears lacking free circulating blood cells. How it clears invading pathogens from its body remains unknown to date. We demonstrate here that amphioxus Branchiostoma belcheri is capable of efficiently eliminating the invading bacterium Escherichia coli from its humoral fluid, and the complement and lysozyme are both involved in the elimination of the invading pathogen. Both the complement and lysozyme act in concert against the invading bacterium, but the complement appears playing a more dominant role than the lysozyme.     

Neuroendocrine regulation of structure and function in the endostyle of amphioxus, Branchiostoma belcheri

Using histological and histochemical methods the structure of the endostyle in amphioxus at different gonadal developmental stages is studied, and the immunocytochemical localization of thyroglobulin (Tg) and thyrotropin (TSH) in the endostyle, Hatschek's pit and brain vesicle is investigated. The results not only confirm the previous report that the endostyle is composed of 6 zones and the cells of zone 5 are thyroid hormone synthesizing cells, but also find the thyroxine synthesis in and secretion from zone 3. In addition, the epithelial cells in Hatschek's pit and the neural cells in brain vesicle are immunopositive for TSH, and the immunoactivity is correlated with gonadal cycle. The present study may provide morphological proof for the hypothesis that the secretory activity of thyroid cells is regulated by TSH from Hatschek's pit and brain vesicle.     

以人工海水和天然海水饲养的文昌鱼生长发育的比较

将 2 0 0尾文昌鱼随机分为 2组 (每组 10 0尾 ) ,分别以人工海水和天然海水在实验室内进行饲养 ,以实验前后文昌鱼的体长、体质量、成活率和性腺直径作比较 ,初步评价人工海水对文昌鱼生长发育的影响 ,并在其生殖季节 ,观察其产卵、排精的情况 .结果表明 :两种不同海水饲养的文昌鱼其成活率、平均体质量及平均性腺直径均无显著差异 ,2组文昌鱼性腺发育基本同步 .但天然海水饲养的文昌鱼体长的增长则明显大于人工海水组 .     

Localization and expression of LHβmRNA in the brain and Hatschek's pit of amphioxus, Branchiostoma belcheri

To determine whether gonadotropin-like substance in the brain and Hatschek' s pit of amphioxus issynthesized by the tissue in situ or transported from other tissue, a histochemical study was carried out by in situ hy- bridization using digoxigenin (DIG)-labelled LHβRNAprobes. The results showed that LHβmRNA expressed in the nerve cells of brain and the epithelial cells of Hatschek' s pit, thus providing new evidence for the homology of pituitary of ver- tebrates with Hatschek' s pit and the functional evolution of gonadotropin.     

Immunolocalization of aromatase, estrogen and estrogen receptor α and β in the epithelium of digestive tract and enteric neurons of amphioxus (Branchiostoma belcheri )

Immunohistochemical localization of aromatase, estrogen and estrogen receptor in the digestive tract and enteric neurons of amphioxus is investigated. It was found that immunoreactive proteins of aromatase, estrogen and ER-α and β are expressed in hepatic diverticulum, epithelial cells of anterior and posterior region of midgut, as well as in enteric neurons, while hindgut showed immunonegative. The results suggest that digestive tract of amphioxus may be able to synthesize estrogen and possess endocrine function, like rat gastric epithelium and enteric neurons in mammals. The present study provides authentic morphological evidence for explaining the action mechanism of estrogen in regulating the digestive function of gut and the functional evolution of estrogen, which has important theoretical significance in amphioxus.     

Distribution of growth hormone-like immunoreactive cells and somatostatin receptors in the nervous system and Hatschek''''s pit of amphioxus, Branchiostoma belcheri

Using immunohistochemical method and double staining technique, the localization of growth hormone (GH) and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus has been investigated. The results showed that the growth hormone-like nerve cells and endocrine cells as well as three subtypes of somatostatin receptors exist in the nervous system and Hatschek s pit, and GH-like nerve cells and endocrine cells co-exist with three subtypes of somatostatin receptors in the brain vesicle and Hatschek s pit. It is suggested that a primitive control system of inhibitory growth hormone secretion in Hatschek s pit could have been developed in amphioxus, as in vertebrates. The present study provides new evidence for the endocrinology and the evolution of Hatschek's pit.     

Distribution of α-MSH-like immunoreactive cells in the nervous system, Hatschek''''s pit and other tissues of amphioxus, Branchiostoma belcheri

Immunohistochemical localization of a-melanocyte-stimulating hormone (α-MSH) in the nervous system, Hatschek's pit and other tissues of amphioxus (Branchiostoma belcheri) was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek's pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek's pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek's pit and for the origin and evolution of vertebrate adenohypophysis.