甘蓝夜蛾和八字地老虎肌动蛋白基因的克隆与序列分析

肌动蛋白是细胞骨架微丝的主要组成成分,在肌肉收缩、细胞骨架形成、细胞移动等方面起重要作用。以鳞翅目夜蛾科昆虫甘蓝夜蛾Mamestra brassicae L.和八字地老虎Agrotis c-nigrum 3龄幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到2种昆虫的肌动蛋白的cDNA序列,甘蓝夜蛾肌动蛋白的cDNA序列含有1441个碱基,而八字地老虎肌动蛋白的cDNA序列含有1411个碱基。2种昆虫的该基因的cDNA序列均包括1个1131个碱基的开放阅读框,编码1个含376个氨基酸的蛋白。甘蓝夜蛾肌动蛋白分子量约为41.8kDa;八字地老虎肌动蛋白分子量约为41.9kDa。Prosite软件分析结果表明,甘蓝夜蛾和八字地老虎肌动蛋白氨基酸序列中存在3个肌动蛋白特征片段。GenBank数据库搜索及序列比对结果表明,甘蓝夜蛾肌动蛋白属于肌肉特异型肌动蛋白,八字地老虎肌动蛋白属于细胞质特异型肌动蛋白。2个基因的cDNA序列已经登录GenBank并获得登录号,甘蓝夜蛾肌动蛋白cDNA序列登录号为EU035314,八字地老虎肌动蛋白cDNA序列登录号为EU035315。利用RT-PCR技术在八字地老虎4龄、5龄、6龄幼虫、蛹期4个不同发育阶段和6龄期的肠道、体壁、脂肪体3种不同组织中都检测到了肌动蛋白基因在mRNA水平的表达。     

小地老虎β-微管蛋白基因cDNA序列的克隆、序列分析和表达检测

β-微管蛋白在昆虫生长发育、信号传导、抗药性等方面具有重要作用.本研究以小地老虎Agrotis ypsilon(Rottemberg)3龄幼虫为材料,利用RT-PCR、cDNA末端快速扩增技术(RACE)克隆得到小地老虎β-微管蛋白基因的cDNA序列,命名为AgTubB(GenBank登录号:JN029962),并检测...     

二化螟β1微管蛋白基因cDNA序列的克隆与序列分析

微管是真核生物体内分布最为广泛的一类蛋白,是由a-和β-两种不同的微管蛋白组成的异源二聚体.微管参与许多细胞功能,如细胞形态发生、细胞生长和分裂等.以二化螟3龄幼虫为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),扩增得到该虫的β微管蛋白基因的cDNA序列一条.cDNA序列含1 862个碱基,开放读码框1 344个碱基,编码氨基酸447个,分子量约为50.2kD,等电点4.82.氨基酸序列中1～4个氨基酸MREI为β微管蛋白转录后调控信号,是微管蛋白特异片段;氨基酸序列的140～146GGGTGSG位存在一个微管蛋白标志信号片段.序列比对表明,克隆的β微管蛋白基因与其他昆虫的β微管蛋白基因在核苷酸和氨基酸水平上都是高度同源的,与家蚕Bombyx moriβ1微管蛋白的氨基酸序列同源性达到99.1%,与烟草天蛾β1微管蛋白的氨基酸序列同源性达到98.7%,与果蝇Drosophila melanogasterβ1微管蛋白的氨基酸序列同源性达到96.9%.该基因cDNA序列已经登录Genbank并获得登录号为EU429675.     

小地老虎UV视蛋白基因的克隆及序列分析

视蛋白是感光物质的重要组成成分,是动物感知周围光环境的重要途径之一。本文以小地老虎(Agrotis ypsilon)3日龄成虫为材料,利用RT-PCR和RACE末端扩增技术克隆得到小地老虎UV视蛋白基因的cDNA序列。序列分析表明,小地老虎视蛋白基因的cDNA序列1 632 bp,包括一个1 140 bp的完整开放阅读框架,编码379个氨基酸,理论蛋白分子量(Mw)41.50 ku,等电点(pI)7.56。GenBank登录号为JN185654。UV视蛋白包括7个跨膜拓扑结构和一个G蛋白偶联受体家族,第107位赖氨酸与UV视蛋白的紫外敏感性有重要关系。同源性分析显示,小地老虎UV视蛋白基因与其他昆虫的UV视蛋白基因具有较高同源性。本研究对深入探究UV视蛋白在动物夜行生活中的作用具有重要意义。     

小地老虎β-N-乙酰葡萄糖胺糖苷酶基因cDNA序列的克隆及mRNA组织特异性表达

β-N-乙酰葡萄糖胺糖苷酶是昆虫几丁质代谢中一种关键酶,在昆虫的多种生理活动中发挥重要作用。本文以小地老虎Agrotis ipsilon Hufngel预蛹期幼虫为材料提取总RNA,利用RT-PCR和RACE技术,扩增得到其β-N-乙酰葡萄糖胺糖苷酶基因的cDNA序列。该序列含有2701个碱基,包括一个1788个碱基的开放阅读框,编码一个含595个氨基酸的蛋白,分子量约为68.3ku,等电点为5.48。推导得到的氨基酸序列与其他昆虫,尤其是鳞翅目昆虫的β-N-乙酰葡萄糖胺糖苷酶高度同源。基因cDNA序列已经登录GenBank并获得登录号GU985280。该基因在小地老虎取食期和预蛹期的马氏管、中肠、脂肪体、体壁和去中肠虫体中均含有mRNA水平的表达。     

甘蓝夜蛾几丁质酶基因cDNA序列的克隆与序列分析

昆虫几丁质酶在害虫生物防治中具有很大的发展潜力。以甘蓝夜蛾Mamestra brassicae L.预蛹期幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),扩增得到其几丁质酶的cDNA序列。该序列含有2826个碱基,包括1个1689个碱基的开放阅读框,预测编码1个含562个氨基酸的多肽,分子量约为62.6kDa,等电点为5.30。推导得到的氨基酸序列含有2个N-位糖基化位点,22个O-位糖基化位点,氨基酸序列与其他昆虫,尤其是鳞翅目昆虫的几丁质酶高度同源。获得的甘蓝夜蛾几丁质酶基因cDNA序列已经登录GenBank并获得登录号FJ436415。     

棉铃虫β-微管蛋白基因cDNA序列的克隆与序列分析

β-微管蛋白是构成细胞骨架的重要组成性蛋白,对昆虫的蜕皮、器官形成等生长发育阶段均能产生重要影响。本文以棉铃虫Helicoverpa armigera(Hübner)3日龄成虫为材料,利用RACE末端扩增技术克隆得到棉铃虫的β-微管蛋白基因的cDNA序列。序列分析表明:棉铃虫β-微管蛋白基因的cDNA序列包含1775个碱基,包括一个1347个碱基的开放阅读框,编码448个氨基酸组成的多肽。GenBank登录号:JF767013。同源性分析表明,棉铃虫的微管蛋白基因与本研究所比对其它昆虫的β-微管蛋白基因具有高度的同源性,达到90%左右。本研究克隆得到棉铃虫的β-微管蛋白基因的cDNA序列,对进一步深入研究该基因功能有重要意义。     

八字地老虎和粘虫蜕皮激素接受子3(HR3)基因cDNA序列的克隆与序列分析

蜕皮激素接受子3(hormone receptor 3,HR3),是一种蜕皮调节转录因子,调控蜕皮过程中相关基因的表达,是蜕皮级联反应中的关键因子。本文以八字地老虎Agrotisc-nigrumL.和粘虫Mythimna separata Walker预蛹期幼虫为材料,分别提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到2种昆虫蜕皮激素接受子3(HR3)的5′非编码区和完整开放读码框在内的cDNA序列,其中八字地老虎的HR3 cDNA序列含有1729个碱基,包括一个1533个碱基的开放阅读框,编码一个含510个氨基酸的蛋白,分子量约为57.5ku。粘虫的HR3cDNA序列含有1743个碱基,包括一个1536个碱基的开放阅读框,编码一个含511个氨基酸的蛋白,分子量约为57.9ku。这2种昆虫HR3 cDNA序列推导的氨基酸序列均具有昆虫核受体超家族特征性结构域,与其他昆虫,尤其是鳞翅目昆虫的蜕皮激素接受子3的氨基酸序列高度同源。获得的基因cDNA序列已经登录GenBank并获得登录号,八字地老虎HR3登录号为GU188853,粘虫HR3登录号为GU188854。     

赤拟谷盗β1-微管蛋白cDNA基因的克隆及序列分析

本研究利用反转录聚合酶链式反应（RT-PCR）和快速扩增cDNA末端（RACE）技术对赤拟谷盗Tribolium costaneum体内β1-微管蛋白（β1-tubulin）基因进行了克隆，获得的基因（EU555191）全长为1573bp，包含1344bp的完整开放阅读框（ORF）。根据该基因推导出447个氨基酸序列，理论分子量约为50KD，等电点为4．68，该序列含有2个氨基酸保守区：NNWAKGHY和RKAFLHWYTGEGMDEMEMEFTE，并且在该基因的5’端启动子调控区发现TATA-box保守基因序列。系统发育关系研究表明：本研究扩增出的基因与其它昆虫β-tubulin基因序列有较高的同源性，达90％左右。     

甜菜夜蛾烟碱型乙酰胆碱受体β1亚基基因的克隆与序列分析

烟碱型乙酰胆碱受体是昆虫体内重要的神经受体,同时也是杀虫剂作用靶标.从甜菜夜蛾Spodoptera exigua3龄幼虫体内提取总的RNA,经过反转录,利用RT-PCR获得了烟碱型乙酰胆碱受体6个α和1个β亚基基因的cD-NA序列片段,并利用cDNA末端快速扩增技术(RACE)获得了β亚基基因的cDNA序列全长.该基因命名为SenAChRβl,其长度为2231个碱基,含有一个1575个碱基的开放读码框,开放读码框编码524个氨基酸残基,预测的分子量为60 kDa.推导得到的氨基酸序列与其它昆虫特别是鳞翅目昆虫的烟碱型乙酰胆碱受体β亚基具有高度的同源性,并具有典型的烟碱型乙酰胆碱受体β亚基特征化位点.     

Spatial distribution of mDLG6 mRNA in embryonic and adult mouse brain

We have isolated mouse DLG6 (mDLG6) cDNA clones by RT-PCR and then by using the RT-PCR products to screen a mouse brain cDNA library. The deduced amino acid sequence of mDLG6 shows 79.2% and 82.7% overall identity to human (hDLG6) and rat DLG6 (rDLG6), respectively. In situ hybridization revealed that mDLG6 mRNA is predominantly expressed in embryonic and adult brain.     

Isolation of bovine liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase cDNA: bovine liver and heart forms of the enzyme are separate gene products.

In order to ascertain whether the heart and liver forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were products of two different genes or arose via alternative splicing of a single gene, the bovine liver cDNA of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a lambda gt10 phage library and its sequence compared with that of bovine heart cDNA. The deduced amino acid sequence of the bovine liver cDNA was also compared with the amino acid sequence of the human and rat liver phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme. The bovine liver cDNA codes for a protein that has 81.6% amino acid identity with the bovine heart form and 97.0 and 98.3% identity with the rat and human liver forms of the enzyme, respectively. Comparison of the nucleotide sequences of the two bovine cDNAs and their deduced amino acid sequences demonstrates that while there is conservation of the active sites of liver/muscle and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases they are encoded by different genes.     

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.     

黑子南瓜甘油-3-磷酸酰基转移酶基因的克隆及序列分析

依据国外报道的南瓜甘油－３－磷酸转酰酶（ＧＰＡＴ）基因的ｃＤＮＡ序列合成相应引物,用ＲＴ－ＰＣＲ技术,成功地分离了黑子南瓜（Ｃｕｃｕｒｂｉｔａｆｉｃｉｆｏｌｉａ）ＧＰＡＴ基因的ｃＤＮＡ片段,并亚克隆到了ｐＧＥＭ－Ｔ载体系统的多克隆位点上,序列分析表明黑子南瓜ＧＰＡＴ基因的ｃＤＮＡ序列及递推的氨基酸序列与南瓜（Ｃｕｃｕｒｂｉｔａｍｏｓｃｈａｔａ）相比分别具有９８％和９６５％的同源性。在１１８８ｂｐ中有２２个核苷酸发生变化,导致１３个氨基酸的改变     

吸血诱导的埃及伊蚊海口株后期胰蛋白酶基因测序及与美国株同源性比较

应用逆转录-聚合酶链式反应（RT-PCR）技术从吸血后24 h埃及伊蚊海口株总RNA中扩增出了后期胰蛋白酶编码区cDNA序列。采用自动DNA分析仪进行序列分析,并与已知埃及伊蚊美国株后期胰蛋白酶基因及推导的氨基酸序列进行了同源性比较。结果表明：埃及伊蚊海口株后期胰蛋白酶基因序列与美国株同源性达98%,有11个碱基发生变异;氨基酸同源性达99%,仅有3个氨基酸发生变异,但与催化位点密切相关的氨基酸及N末端氨基酸序列完全一致。以上结果显示,埃及伊蚊胰蛋白酶不同地理株间存在微小的差异。     

Mouse liver cytochrome P-450 (P-450IIIAM1): its cDNA cloning and inducibility by dexamethasone.

A full-length cDNA complementary to mouse liver mRNA coding for one of the cytochromes P-450 (P-450) in the P-450IIIA family, namely P-450IIIM1, was isolated and completely sequenced. The sequence of this cDNA clone, pMDex13, revealed that it encoded a polypeptide of 504 deduced amino acid residues (Mr = 57,853). The deduced amino acid sequence showed 87.3 and 84.9% identity with rat P-450IIIA1 and P-450IIIA2, respectively. The NH2-terminal 24 amino acid sequences of P-450IIIAM1 were completely identical with purified mouse P-450UT protein. RNA blot analysis showed that mRNA content of hepatic P-450IIIAM1 was remarkably increased by treatment of mice with dexamethasone.     

Tubulin Genes in the Algal Protist Euglena gracilis

ABSTRACT Alpha- and beta-tubulin cDNA were selected from a Euglenaλgt11 expression library, recloned and either sequenced (α-tubulin cDNA) or hybridized to Euglena RNA and DNA (α- and β-tubulin cDNA). RNA for hybridization was extracted at 30 minute intervals after flagellar amputation and quantitated for cDNA binding. Unlike previous reports on most other flagellates, no net increase in either α- or β-tubulin RNA could be detected during regeneration—suggesting steady state or constitutive tubulin RNA synthesis. Incubation of the cDNA with genomic DNA after restriction digestion produced patterns of hybridization consistent with the presence of one to two kinds each of the α- and β-tubulin genes. The deduced amino acid sequence of the α-tubulin cDNA was more than 90% identical to the α-tubulins of Trypanosoma, Chlamydomonas, Naegleria, Tetrahymena and higher plants. The carboxy terminus of the α-tubulin cDNA and the previously sequenced β-tubulin of Euglena showed greatest identity to the carboxy terminus of the tubulins from Trypanosoma brucei. The sequence data for α and β-tubulins of Euglena provides direct evidence for the similarity of two gene products from euglenas and trypanosomes and adds support to earlier suggestions that these organisms are phylogenetically related.