鲤鱼(Cyprinus carpio)生长激素基因克隆及原核表达

采用逆转录—聚合酶链式反应（ＲＴ－ＰＣＲ）方法,从鲤鱼脑垂体总ＲＮＡ中扩增出编码鲤鱼生长激素（ＧＨ）成熟肽基因序列．定向克隆至质粒ｐＵＣ１８,克隆的鲤鱼ＧＨｃＤＮＡ不含信号肽序列并以新的起始密码子ＡＴＧ取代鲤鱼ＧＨｃＤＮＡ第１个密码子ＴＣＡ．序列分析表明,与Ｋｏｒｅｎ报道的鲤鱼ＧＨｃＤＮＡ相比有两个碱基差异,但推断的氨基酸序列完全一致．将鲤鱼ＧＨｃＤＮＡ定向克隆至原核表达载体ｐＢＶ２２０,构建成重组鲤鱼ＧＨ基因表达载体ｐＢＶｃＧＨ８．ＳＤＳ－ＰＡＧＥ和薄层扫描分析表明：经４２℃诱导,ｐＢＶｃＧＨ８在大肠杆菌中可表达一分子量约２２０００的特异蛋白,表达量占细胞总蛋白的２９．２％．该基因重组的鲤鱼ＧＨ添加到饲料中投喂罗非鱼,证实有明显的促进生长作用     

RT—PCR测定mRNA的荧光定量分析

利用反转录毛细管ＰＣＲ技术,可合成代表特异ｍＲＮＡ的双链ＤＮＡ。在一定循环数内,ＰＣＲ产物的量与其模板ｃＤＮＡ即反转录中相应ｍＲＮＡ的浓度有关。溴乙锭嵌入ＤＮＡ双螺旋后,其荧光量子产率大为增加,因此可通过利用处在适当ＰＣＲ循环数中的ｃＤＮＡ浓度与在优选的激发光谱发现射波长下其荧光强度的线性关系,计算出所检测的ｍＲＮＡ表达量。     

cDNA3‘端代表差异显示分析

根据真核ｍＲＮＡ３’端一般含Ｐｏｌｙ（Ａ）的原理,可使用共同引物将不同的ｍＲＮＡ反转录成ｃＤＮＡ,然后设计特殊引物进行反转录ＰＣＲ,或者将限性酶切与呱转录ＰＣＲ偶联使用,均可获得对应于ｍＲＮＡ３’端的ｃＤＮＡ３’端代表扩增子。     

鳄梨冷藏过程中体外转译和基因表达的研究

鳄梨果实经低温贮藏一定时期,从中提取ＲＮＡ,进行体外转译和同源探针杂交分析,以研究冷藏过程中特写基因的表达时期和水平。并用纯化出的冷藏果实的ｍＲＮＡｓ,构建其ｃＮＤＡ文库。对文库作了初步筛选,２４个含ｃＤＮＡ插入片段的菌落能差示地与贮藏前和７℃贮藏的ｍＲＮＡ合成的探针杂交。３个纤维素酶ｃＤＮＡ能很好杂交,２个与鳄梨乙烯形成酶ｃＤＮＡ有同源性。     

原位杂交检测大鼠前庭代偿中GAP-43 mRNA水平

用ＤＩＧ标记的ＧＡＰ－４３ｃＤＮＡ为探针,以大鼠海马切片作阳性对照,使用原位杂交方法检测了大鼠迷路损毁５,１２,２０和３０ｄ后前庭核区ＧＡＰ－４３ｍＲＮＡ水平的变化,结果表明,迷路损毁后前庭核区ｍＲＮＡ水平升高,原位杂交的应用,为前庭代偿中轴突发芽,突触重组的神经可塑性研究打下了方法学基础。     

cDNA3′端代表差异显示分析

根据真核ｍＲＮＡ３′端一般含Ｐｏｌｙ（Ａ）的原理,可使用共同引物将不同的ｍＲＮＡ反转录成ｃＤＮＡ,然后设计特殊引物进行反转录ＰＣＲ,或者将限制性酶切与反转录ＰＣＲ偶联使用,均可获得对应于ｍＲＮＡ３′端的ｃＤＮＡ３′端代表扩增子。比较不同条件下的ｃＤ－ＮＡ３′端代表扩增子,可以获得两种或多种细胞中ｍＲＮＡ的表达差异谱,并分离、克隆差异表达的基因序列     

cDNA3′端代表差异显示分析

根据真核ｍＲＮＡ３′端一般含Ｐｏｌｙ（Ａ）的原理,可使用共同引物将不同的ｍＲＮＡ反转录成ｃＤＮＡ,然后设计特殊引物进行反转录ＰＣＲ,或者将限制性酶切与反转录ＰＣＲ偶联使用,均可获得对应于ｍＲＮＡ３′端的ｃＤＮＡ３′端代表扩增子。比较不同条件下的ｃＤ－ＮＡ３′端代表扩增子,可以获得两种或多种细胞中ｍＲＮＡ的表达差异谱,并分离、克隆差异表达的基因序列 。     

迷路损伤增强大鼠前庭内侧核生长相关蛋白mRNA水平

前庭代偿是研究神经可塑性的一个理想模型。生长相关蛋白（ＧＡＰ－４３）在神经再生和突触重组中起重要作用。用ＤＩＧ标记的ＧＡＰ－４３ ｃＤＮＡ片段作探针进行原位杂交,检测了大鼠迷路损伤５、１２、２０和３０ｄ后前庭内侧核ＧＡＰ－ｍＲＮＡ表达的变化。结果表明,迷路损毁后两侧前庭内侧核ＧＡＰ－４３ｍＲＮＡ的水平以不同的幅度和时程明显升高。这一结果表示,ＧＡＰ－４３ｍＲＮＡ水平的提高可能与前庭代偿中突触重组和     

簇毛麦低拷贝cDNA序列的克隆和鉴定

从簇毛麦叶片中提取总ＲＮＡ,进一步分离ｍＲＮＡ。以ｍＲＮＡ为模板反转录合成ｃＤＮＡ,两端经Ｔ４ＤＮＡ多聚酶修平后加ＥｃｏＲ１接头分子,连接于质粒ｐＧＥＭ－７Ｚｆ（＋）的ＥｃｏＲ１克隆位点,转化大肠杆菌ＪＭ１０３菌株建立了ｃＤＮＡ文库。用ＰＣＲ扩增重组质粒的ｃＤＮＡ插入序列,用＾３２Ｐ标记后分别与ＨｉｎｄⅢ或ＸｂａⅠ酶切的小麦－黑麦附加系ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交。根据其杂交结果,目前已鉴定出４     

mRNA差异显示技术在动物早期胚胎发育相关基因研究中的应用

动物从卵裂开始的早期胚胎发育始终伴随着ｍＲＮＡ不断的合成与降解。最初受着受精卵内母体ｍＲＮＡ的控制。当这些ｍＲＮＡ在发育过程中逐渐被降解时,有些动物（如小鼠）在比较早的时期已有新基因的转录;而有些动物（如鱼类、两栖类）迟到囊胚中期以后,合子核的基因才开始转录新的ｍＲＮＡ。ｍＲＮＡ这种有规律的代谢活动控制着细胞的分化、胚层的形成以及模式形成（ｐａｔｅｒｎｆｏｒｍａｔｉｏｎ）。所有这些ｍＲＮＡ水平上的变化都可以用ｍＲＮＡ差异显示法（ｍＲＮＡｄｉｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ）检测出来并进而获得与早期胚胎发育有关的基因。ｍＲＮＡ差异显示法是由Ｌｉａｎｇ和Ｐａｒｄｅｅ于１９９２年建立起来的,用于显示两种不同组织之间ｍＲＮＡ差异的方法。基于绝大多数ｍＲＮＡ有一个ｐｏｌｙ（Ａ）尾巴,选用一个较特殊的３’端引物─５’Ｔ１１ＭＮ３’,其中Ｍ可以是ｄＡＴＰ,ｄＣＴＰ,ｄＧＴＰ之一,而Ｎ可以是ｄＡＴＰ,ｄＴＴＰ,ｄＣＴＰ,ｄＧＴＰ之一,进行逆转录时,１／１２的ｍＲＮＡ可被逆转录为ｃＤＮＡ。这种ｃＤＮＡ第一链被用来ＰＣＲ扩增,所使用的３’端引物与逆转录引物相同,而５’端引物为１０个碱基的一段寡核苷酸,这类任意（ａｒｂｉｔｒａｒ     

丘脑下部促黄体素释放激素类似物(LRH-A)的作用机制 Ⅲ.对罗非鱼脑垂体促性腺激素分泌细胞超微结构的影响

前文(方永强等,1981)探讨了罗非鱼脑垂体促性腺细胞的生理活动与雌鱼性腺发育不同时期的相互关系,对比注射LRH-A后2、4和10小时对促性腺细胞的影响。根据促性腺细胞核的形态和细胞质中分泌颗粒数量的变化,以及DNA、RNA、酸碱性蛋白和酸碱性磷酸酶等一系列反应,初步阐明了LRH-A的作用机制与细胞核和细胞质的代谢变化有关。在此基础上,本文较详细观察了LRH-A对垂体促性腺细胞超微结构的影响,目的在于从亚细胞的水平上,进一步了解其作用机制。     

HORMONAL CONTROL OF THE VITELLOGENESIS IN THE JAPANESE OAK SILKWORM, ANTHERAEA YAMAMAZ (LEPIDOPTERA: SATURNIIDAE)

Abstract Effects of ecdysteroid and juvenile hormone (JH) on vitellogenesis of the Japanese oak silkworm, Antheraea yamami are reported in this article. After topical treatment with 20-hydroxyecdysone alone or JH analog (i.e. methoprene) alone and combined treatment with these two chemicals, vitellogenin (Vg) titers in the fat body and haemolymph at the pupal stage were mostly higher than those of the control, indicating that both ecdysteroid and JH exerted a promoting effect on the synthesis of Vg. In contrast, the Vg uptake was markedly inhibited by JH while stimulating effect of the ecdysteroid could be shown that vitellin (Vt) titer in the ovary was lower after methoprene treatments, but higher after 20-hydroxyecdyson treatments. Meanwhile, effects of these two hormones on Vg synthesis in the fat body were also tested with the incubation in vitro with Grace medium containing H-leucine and the hormones. The results demonstrated that Vg synthesis was stimulated after treating with methoprene alone or 20-hydroxyecdysone alone and combined treating with these two chemicals, and particularly ecdysteroid had more marked positive effect. To comprehensively concluded our results, it could be regarded that ecdysteroid play the main role in the regulation of vitellogenesis for the Japanese oak silkworm.     

早熟素Ⅱ处理粘虫蛾对两性吸引的影响

本文首次报道早熟素Ⅱ对鳞翅目重要害虫粘虫Mythimna separata Walker的作用。试验证明羽化24小时以内未交配的粘虫蛾(雌雄比为1:1)与10微克/平方厘米的早熟素接触24小时后即明显地降低两性吸引的能力;雌雄比为2:1时,处理组的交配率比对照组降低60—70%。不同的配对(正常雄×正常雌、正常雄×处理雌、正常雌×处理雄、处理雌×处理雄)证明早熟素Ⅱ对雄、雌蛾均有影响,但对雄蛾的影响更大。已知昆虫咽侧体(CA)分泌的保幼激素(JH)对某些昆虫成虫的两性吸引有调控作用(Barth,1961;1962),本文对此进行了探讨,对早熟素处理后的雌、雄蛾咽侧体逐日进行了测量,它们都比对照小,雄蛾差异更大。雄蛾CA的组织切片也表现出明显的异常:细胞核变形,染色体颗粒凝聚、出现大量空斑,腺体腔内分泌物较少等。同位素标记甲硫氨酸测定离体CA合成JH的结果表明:早熟素处理的雄蛾CA合成JH能力下降,在正常情况下出现高峰的第四天左右差异更为明显,雌蛾也有降低的趋势。     

天蚕卵黄发生的激素调控

报告了蜕皮激素和保幼激素对天蚕Antheraea yamamai卵黄发生的调控作用。当单独以20－羟基蜕皮酮或保幼激素类似物methoprene处理,以及同时用这两种激素处理天蚕蛹时,蛹期脂肪体和血淋巴中卵黄原蛋白（Vg)含量明显高于对照,即二对Vg的合成起促进作用。然而,卵巢中卵黄蛋白（Vt)含量则因激素种类而异,以保幼激素处理时明显低于对照,以20－羟基蜕皮酮处理则反之,即前抑制卵巢对Vg的摄取,而后则起促进作用。离体培养脂肪体并以激素处理的结果表明,20－羟基蜕皮酮和methoprene均能促进Vg合成,但前作用更。综合考虑上述结果可以认为蜕皮激素对该蚕的卵黄发生起主要调控作用。     

FUNCTIONAL REDUCTION OF THE SOUTHERN MINKE WHALE (BALAENOPTERA ACUTOROSTRATA) TESTIS DURING THE FEEDING SEASON

Seasonal variation in reproductive activity in the male southern minke whales was investigated. The animals were killed and collected during the feeding season (December-March) in the Antarctic Ocean in the eastern part of Area III (35°E-70°E, south of 60°S) and Area IV (70°E-130°E, south of 60°S). Blood samples, testes, epididymides, and vasa deferentia were collected from 62 males, which had testes weighing over 400 g each. Changes in testicular morphology and plasma testosterone, estradiol-17β and LH concentrations measured by enzyme immunoassay were investigated. Reduction of testicular function associated with different capture periods was found in the summer season. From December to March, body length and body weight remained steady, but decreases in testicular weight, epididymal weight, and testicular volume were found in February. During the same period, plasma testosterone concentration also declined. A significant decrease in the number of germ cells continued during the period of feeding season. An increase in area of seminiferous tubule tended to proceed in a number of germ cells. These changes reflected the percentage of spermatozoa in the vasa deferentia ; in particular, motile spermatozoa were observed in December. Based on morphological observation, spermatogenesis had also declined. These results indicate regulation of testicular function by testosterone, as in terrestrial mammals. A gradual decrease of testicular function occurred during December-January.     

THE GENETIC BASIS OF MALE POTENCY IN VOLVOX CARTERI F. NAGARIENSIS (CHLOROPHYCEAE)1,2

Sexual males of Volvox carteri Stein f. nagariensis Iyengar secrete a hormone which induces sexual rather than asexual development in the next generation. Male recombinants from the original strains isolated from Japan showed an increase in potency of up to 100 times that of the original male clone, with later descendants showing potencies ranging from 100 times to 1/1000 the potency of this male. Potency is defined as the extent to which the hormone produced in 1 ml of medium by one parental spheroid containing 16 male spheroids can be diluted and retain its capacity to induce sexual differentiation. This is recorded as the percent of female response in the tube of limiting dilution, asexual females of the normal female strain being used as assay inoculum in a 1 in 10 dilution series of samples of male fluid containing the sexual hormone. Investigation of the genetic basis for potency in this species included selection of high and low potency male clones and crossing with sib females or backcrossing with original female. Results of the various crosses are interpreted as multigenic system not linked with sex, with more than one genotypic class per dilution level and with more than one genotypic class per apparent phenotypic class. Possible genic interactions are described in their relevance to the biosynthesis of a glycoprotein molecule, the sexual hormone having been characterized as a glycoprotein by Starr and Jaenicke (18).     

CHARACTERIZATION AND FUNCTIONAL STUDY OF A PUTATIVE JUVENILE HORMONE DIOL KINASE IN THE COLORADO POTATO BEETLE Leptinotarsa decemlineata (Say)

Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH degradation. In the present article, a putative JHDK cDNA (LdJHDK) was cloned from the Colorado potato beetle Leptinotarsa decemlineata. The cDNA consists of 814 bp, containing a 555 bp open reading frame encoding a 184 amino acid protein. LdJHDK reveals a high degree of identity to the previously reported insect JHDKs. It possesses three conserved purine nucleotide‐binding elements, and contains three EF‐hand motifs (helix‐loop‐helix structural domains). LdJHDK mRNA was mainly detected in hindgut and Malpighian tubules. Besides, a trace amount of LdJHDK mRNA was also found in thoracic muscles, brain–corpora cardiaca–corpora allata complex, foregut, midgut, ventral ganglia, fat body, epidermis, and hemocytes. Moreover, LdJHDK was expressed throughout all developmental stages. Within the first, second, and third larval instar, the expression levels of LdJHDK were higher just before and right after the molt, and were lower in the intermediate instar. In the fourth larval instar, the highest peak of LdJHDK occurred 56 h after ecdysis. Ingestion of double‐stranded RNA (dsRNA) against LdJHDK successfully knocked down the target gene, increased JH titer, and significantly upregulated LdKr‐h1 mRNA level. Knockdown of LdJHDK significantly impaired adult emergence. Thus, we provide a line of experimental evidence in L. decemlineata to support that LdJHDK encodes function protein involved in JH degradation.     

草鱼生长激素非竞争式酶联免疫吸附测定法的建立及鉴定

应用草鱼生长激素（ｇｃＧＨ）单克隆抗体及多价兔抗血清建立了草鱼ＧＨ非竞争式酶’联免疫吸附测定ＥＬＩＳＡ系统。用正辛酸法对腹水单抗进行了分离纯化,获得了高纯度的单抗制备物。聚丙烯酸胺凝胶电泳表明纯化的单抗由分子量分别为５５ｋＤ和２５ｋＤ的两条蛋白带组成。用纯化单抗铺底,用兔抗血清作后续抗体建立了一种测定草鱼ＧＨ的非竞争式双抗夹心ＥＬＩＳＡ方法。交叉试验表明该测定系统只与草鱼ＧＨ和基因重组鲤生长激素（ｒｃＧＨ）具有剂量依存的结合反应,而与大马哈鱼生长激素（ｓＧＨ）、牛生长激素（ｂＧＨ）、大马哈鱼促性腺激素（ｓＧｔＨ）、及黑鲢促性腺激素（ｂｓｃＧｔＨ）等均无交叉反应。该 ＥＬＩＳＡ方法的灵敏度可达０．８ｎｇ／ｍｌ,组内变异系数为 ５．９ ％,组间变异系数为７．６％,回收率达９０％以上。初步应用表明,鲤和团头鲂垂体抽提液、草鱼血清、鲤血清及鲫血清在该测定系统中有剂量依存的反应曲线,而大口鲶、黄颡鱼、中华鲟及黄鳝鱼垂体抽提液及大口鲶、胡子鲶和罗非鱼血清在该测定系统中没有交叉反应。     

气相色谱-质谱法分离鉴定胡桃(Juglans regia)花粉中的甾类激素

用有机溶剂提取胡桃花粉中的甾类化合物,以70％甲醇除掉脂类杂质,然后将提取出来的甾类化合物衍生化,经气相色谱分离,质谱仪鉴定,搞清胡桃花粉中含有雌二醇、谷甾醇等甾类激素;植物雄配子体中存在有动物雌性激素。     

蛙类下丘脑—脑垂体—性腺轴的内分泌调节

本文从“下丘脑－脑垂本－性腺”轴方面,综述了蛙类生殖内分泌学研究领域所限得的主要成就和研究进展。对于今后的工作,从理论和生产方面提出了一些看法。