RNAi介导的棉铃虫氨肽酶N基因Haapn1和钙粘蛋白基因Ha_BtR沉默对Cry1Ac毒力的影响

氨肽酶N（aminopeptidase N, APN）和钙粘蛋白（cadherin）是存在于鳞翅目昆虫中肠刷状缘膜囊（brush border membrane vesicles, BBMV）上Bt毒素Cry1A的受体。本实验将棉铃虫Helicoverpa armigera氨肽酶N1基因Haapn1和钙粘蛋白基因Ha_BtR双链RNA（dsRNA）注入棉铃虫4龄幼虫体内, 以研究这两种受体基因沉默后对Cry1Ac毒力的影响。结果表明: 注射dsRNA（1 μg/头）进行基因沉默后, Haapn1 mRNA表达量比注射缓冲液（elution solution, ES）的对照下降了30%~49%, Ha_BtR mRNA表达量下降了30%~37%。注射Haapn1 dsRNA的幼虫在40和70 μg/cm² Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫, 而在 100 和 170 μg/cm² Cry1Ac原毒素处理下两者死亡率无显著差异; Cry1Ac活化毒素以及原毒素对注射Ha_BtR dsRNA幼虫与注射ES幼虫的毒力均无显著差异。当同时注射Haapn1及Ha_BtR dsRNA后, 干扰后的幼虫对Cry1Ac活化毒素和原毒素的敏感性均显著下降。本研究进一步证明了棉铃虫Haapn1和Ha_BtR均是Bt毒素Cry1Ac的功能受体, 这两种受体蛋白共同参与Cry1Ac的毒杀作用过程。该结果也提示, Haapn1或Ha_BtR基因产生突变都可能导致棉铃虫对Cry1Ac产生抗性。     

菲律宾实蝇生物学特性研究

菲律宾实蝇Bactrocera (Bactrocera) philippinensis (Drew & Hancock)是重要的检疫性有害生物, 严重危害芒果Mangifera indica L.、木瓜Carica papaya L.和菠萝蜜Artocarpus heterophyllus Lam.等水果。本文在室内实验条件下对菲律宾实蝇生物学特性进行了较为系统的研究。结果表明: 菲律宾实蝇在6~18 h羽化率达93.93%。成虫活动、寿命、交配和产卵与温度、光照密切相关。补充营养能显著延长成虫寿命。菲律宾实蝇生长、发育和繁殖的适宜温度为25~30℃。25℃和30℃时平均产卵量分别为627.35粒和652.57粒。发育起点温度和世代有效积温分别为14.31℃和450.43日·度。温度与发育历期呈显著的负相关（r=-0.9005）。本研究为菲律宾实蝇的检疫处理和田间防治技术研究提供了重要的基础资料。     

异色瓢虫成虫体型及体内脂肪含量对其耐寒能力的影响

为明确异色瓢虫Harmonia axyridis (Pallas)成虫体型及体内脂肪含量对其耐寒能力的影响, 本研究在2种发育温度(18℃和25℃)下通过表型可塑性诱导获得了不同体型的异色瓢虫成虫, 测定了成虫体型大小、体内脂肪含量、过冷却点(supercooling point, SCP)及其在恒定低温(constant low temperature, CLT)和变温(fluctuating thermal regime, FTR)下的存活率。结果表明: 较低发育温度(18℃)下的成虫体型比25℃下的明显大(P<0.01), 体内脂肪含量显著高于25℃下的(P<0.01); 且两2种发育温度下成虫体内脂肪含量与虫体干重呈较好的正相关关系。由SCP频次分布图可知, 饲养在18℃下的成虫SCP主要集中在-8 ~ -6℃范围内, 饲养在25℃下的成虫SCP主要集中在-10 ~ -9℃范围内; 且成虫体型大小与过冷却能力呈现负相关关系。经过相同时间低温暴露后成虫的存活率按下列顺序降低: FTR18℃＞FTR25℃＞CLT18℃＞CLT25℃。结果表明低温暴露过程中脂肪贮存对异色瓢虫成虫存活是非常重要的, 但是冷伤害是影响存活的基本因素。     

光滑鳖甲热休克蛋白70基因的克隆及表达

根据GenBank中发表的昆虫热休克蛋白70 (heat shock protein 70, HSP70) 基因的保守序列设计引物,利用PCR结合RACE扩增的方法,获得新疆荒漠昆虫光滑鳖甲Anatolica polita borealis热休克蛋白70基因,命名为APhsp70 (GenBank注册号为EF569673)。测序结果表明,序列长为2 092 bp,由核酸序列推演出的蛋白质分子量为70 kD,含653个氨基酸残基。基因结构分析表明APhsp70不含内含子序列。通过Northern blotting和半定量RT-PCR技术研究昆虫在受到不同温度胁迫时该基因的表达规律,结果表明：在45℃处理时,昆虫体内该基因的表达水平显著上升。并且,当机体受到-5℃低温胁迫并在室温恢复15 min后APhsp70的表达量也显著升高;但是4℃和-10℃低温处理组APhsp70的表达未见明显变化。     

温度对马铃薯甲虫生长发育的影响

为了进一步明确温度对马铃薯甲虫Leptinotarsa decemlineata (Say)生长发育的影响, 在恒温条件, 研究了温度对马铃薯甲虫实验种群生长发育的影响。结果表明：温度对马铃薯甲虫各虫态的发育历期、存活率及种群的繁殖力有显著的影响。发育历期随温度的升高而缩短, 发育速率与温度呈显著的正相关。马铃薯甲虫世代存活率由大到小的顺序为27℃＞23℃＞19℃＞31℃＞15℃。27℃时成虫的产卵量最高, 单雌平均卵量为729.7粒;其次为23℃, 单雌平均卵量为639.2粒。并测得马铃薯甲虫卵、1龄幼虫、2龄幼虫、3龄幼虫、4龄幼虫、幼虫期、蛹期及全世代的发育起点温度分别为9.14, 10.50, 8.17, 10.28, 9.04, 9.59, 10.23和10.90℃, 有效积温分别为73.26, 43.22, 39.23, 34.05, 161.97, 273.02, 100.38和542.58日·度。据此认为温度对马铃薯甲虫实验种群的生长发育、存活和繁殖影响明显。     

黄粉甲抗冻蛋白AFP84a在大肠杆菌中的高效表达及活性检测

为表达和纯化黄粉甲Tenebrio molitor抗冻蛋白, 采用RT-PCR方法扩增得到黄粉甲抗冻蛋白基因afp84a cDNA, 将其连接到pMAL-p2X质粒上, 构建分泌型融合表达载体pMAL-p2X-afp84a, 并在大肠杆菌Escherichia coli TBI中表达; 进一步利用Amylose柱亲和纯化出该重组蛋白, 后利用细菌抗寒性检测重组蛋白的生物活性。结果显示: 融合蛋白含量占总可溶蛋白的40%。SDS-PAGE分析表明, 用MgSO4处理法与超声波细胞破碎法均可使融合蛋白从细胞中释放; 融合蛋白经Amylose柱亲和纯化, Factor Xa因子酶切, 电泳显示获得的目的蛋白呈单一条带。细菌抗寒性检测表明该重组蛋白具有较高的抗冻活性。黄粉甲抗冻蛋白基因afp84a cDNA的克隆、 原核表达为进一步研究抗冻蛋白的性质和应用提供了有用的实验材料。     

小菜蛾对阿维菌素抗性基因的AFLP连锁图谱的构建

世界性害虫小菜蛾Plutella xylostella (L.) 田间种群已对阿维菌素药剂产生了高抗药性。本研究以小菜蛾对阿维菌素的抗性品系、敏感品系和回交群体组建作图家系,再利用AFLP技术构建小菜蛾对阿维菌素抗性基因的连锁图谱。用10组AFLP引物组合对未经药剂处理的小菜蛾成虫DNA选择性扩增,共获得1 044条可区分的DNA条带,271条带具有多态性,χ²检验表明只有123个位点符合1∶1 （P=0.05）,其中的112个构成28个连锁群,其总长度为1 222.7 cM; 用10组AFLP引物对经药剂处理后存活的小菜蛾成虫DNA扩增,所得的DNA条带中有54个条带符合分离比1∶1,经Mapmaker 3.0对多态性位点分析,只有E1M4-15、E1M1-4和E1M4-2标记位点位于同一连锁群,3个AFLP标记与抗性基因的遗传距离分别为0 cM,8.3 cM和13.1 cM。比较分析得出,抗性基因所在的连锁群是第5连锁群。结果显示AFLP连锁图谱对小菜蛾对阿维菌素的抗性监测具有很好的应用潜力。     

龟纹瓢虫广东种群和北京种群的耐热性比较研究

本文以龟纹瓢虫Propylea japonica (Thunberg)的广东和北京两地区种群为材料,对其进行了在不同温度（26℃～36℃）下的发育历期、存活率、种群性比、产卵量的试验观察及对蛹重、幼虫致死高温、亚致死高温预处理、可溶性蛋白质含量的测定等。结果显示：（1）34℃以上温度对两种群龟纹瓢虫的各世代历期均产生了不同程度的影响。广东种群,34℃下的4龄幼虫、成虫产卵前期和世代历期开始延长,在36℃时除1龄幼虫和蛹外,其它虫态的发育历期均比34℃时的显著延长;北京种群,在36℃时,2龄和4龄幼虫的发育历期开始表现为显著延长。雄性比例,在26℃～36℃范围内随着温度上升而增大,至36℃时,两种群成虫均为雄性。从产卵量看,两种群在34℃下急剧下降。（2）在供试条件下,两种群各龄期的存活率,在36℃下开始明显下降,其中对低龄幼虫和卵期影响最大。广东种群卵的孵化率从26℃时的64.3%下降到34℃时的42.3%, 36℃下的则降为20.7%,下降了67.8％;北京种群卵的孵化率从26℃处理的58.0%, 下降到34℃处理的44%,36℃处理仅为12.3%,下降了78.8％。（3）广东种群和北京种群各龄幼虫致死温度相同,分别为1、2龄幼虫44℃、3龄幼虫45℃、4龄幼虫46℃。经39℃亚致死高温预处理150 min后,广东种群和北京种群4龄幼虫在46℃下的存活率分别提高了72%和43.5%。（4）在26℃～34℃之间,随着温度的上升,成虫和4龄幼虫体内可溶性蛋白的含量上升。在常温下生长的4龄幼虫和成虫经39℃、41℃和43℃热激1 h后,4龄幼虫体内可溶性蛋白含量上升,成虫体内可溶性蛋白含量却下降。广东种群4龄幼虫和成虫经39℃热激后可溶性蛋白含量最高,而北京种群经43℃热激后含量最高。本文结果初步明确了龟纹瓢虫对较高温度的忍耐性,发现在36℃下龟纹瓢虫广东种群和北京种群的生长发育和繁殖会受到严重阻碍,对其发育历期、性比、产卵量、存活率等均有不良影响;广东种群和北京种群在31℃下3龄幼虫的发育历期、36℃下世代存活率、经过亚致死高温预处理后的耐热性、热激后虫体内蛋白含量等均存在显著差异。, -bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">热激1 h后,4龄幼虫体内可溶性蛋白含量上升,成虫体内可溶性蛋白含量却下降。广东种群4龄幼虫和成虫经39℃热激后可溶性蛋白含量最高,而北京种群经43℃热激后含量最高。本文结果初步明确了龟纹瓢虫对较高温度的忍耐性,发现在36℃下龟纹瓢虫广东种群和北京种群的生长发育和繁殖会受到严重阻碍,对其发育历期、性比、产卵量、存活率等均有不良影响;广东种群和北京种群在31℃下3龄幼虫的发育历期、36℃下世代存活率、经过亚致死高温预处理后的耐热性、热激后虫体内蛋白含量等均存在显著差异。, -bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA; mso: 'Times New Roman'">热激1 h后,4龄幼虫体内可溶性蛋白含量上升,成虫体内可溶性蛋白含量却下降。广东种群4龄幼虫和成虫经39℃热激后可溶性蛋白含量最高,而北京种群经43℃热激后含量最高。本文结果初步明确了龟纹瓢虫对较高温度的忍耐性,发现在36℃下龟纹瓢虫广东种群和北京种群的生长发育和繁殖会受到严重阻碍,对其发育历期、性比、产卵量、存活率等均有不良影响;广东种群和北京种群在31℃下3龄幼虫的发育历期、36℃下世代存活率、经过亚致死高温预处理后的耐热性、热激后虫体内蛋白含量等均存在显著差异。, -bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">热激1 h后,4龄幼虫体内可溶性蛋白含量上升,成虫体内可溶性蛋白含量却下降。广东种群4龄幼虫和成虫经39℃热激后可溶性蛋白含量最高,而北京种群经43℃热激后含量最高。本文结果初步明确了龟纹瓢虫对较高温度的忍耐性,发现在36℃下龟纹瓢虫广东种群和北京种群的生长发育和繁殖会受到严重阻碍,对其发育历期、性比、产卵量、存活率等均有不良影响;广东种群和北京种群在31℃下3龄幼虫的发育历期、36℃下世代存活率、经过亚致死高温预处理后的耐热性、热激后虫体内蛋白含量等均存在显著差异。     

甜菜夜蛾低龄幼虫取食含Cry1Ac毒素的人工饲料对其生长发育和成虫繁殖的影响

为了更全面地评价转Bt基因作物对甜菜夜蛾Spodoptera exigua (Hübner)（鳞翅目: 夜蛾科）的影响, 在室内研究了甜菜夜蛾低龄幼虫连续6 d取食含不同浓度Cry1Ac毒素的人工饲料后转移到不含毒素的饲料上, 其生长发育与成虫繁殖的变化。结果表明: 低龄幼虫取食含Cry1Ac的饲料6 d内, 校正死亡率随Cry1Ac浓度的升高以及取食时间的延长而升高。与一直取食不含Cry1Ac饲料的对照相比, 取食含Cry1Ac饲料的幼虫体重显著下降; 幼虫历期、预蛹期及雌雄蛹期均显著延长, 但雌雄蛹重均与对照无显著差异; 幼虫化蛹率显著下降, 但羽化率与对照无显著差异; 成虫产卵前期显著延长, 产卵期与对照无显著差异, 仅在5 μg/g处理下较对照显著延长1.3 d; 每雌产卵量以10 μg/g处理最高（719粒）, 但各处理均与对照无显著差异; 雌蛾寿命均显著延长, 而雄蛾寿命仅在最高浓度80 μg/g下显著延长2.4 d。这表明, 尽管含不同浓度Cry1Ac毒素的饲料对甜菜夜蛾低龄幼虫有明显的生长发育抑制作用或导致死亡, 但转移至不含毒素的饲料上取食后, 幼虫能迅速恢复生长, 顺利化蛹、羽化并产卵。因此, 在转Bt基因作物田生长后期, 甜菜夜蛾低龄幼虫取食表达Cry1Ac蛋白的组织若能存活并完成世代发育和繁殖, 这无疑将增加甜菜夜蛾对Bt作物产生抗性的风险, 因此亟需制定Bt作物生态系统中甜菜夜蛾的治理策略。     

家蚕吡哆醛激酶的融合表达与纯化

【目的】吡哆醛激酶（pyridoxal kinase, PLK, EC 2.7.1.35）是维生素B6的关键代谢酶。本研究原核表达家蚕Bombyx mori重组PLK, 为进一步开展家蚕PLK的催化作用机制和表达调控机制的研究奠定基础。【方法】构建家蚕PLK基因融合表达质粒, 转化大肠杆菌Escherichia coli诱导表达, 经Ni²⁺ 亲和层析纯化后, 对融合蛋白的催化活性进行分析。【结果】纯化后的家蚕重组PLK经SDS-PAGE鉴定为单一条带, 比活力为1 800 U/mg, 纯化倍数为40倍。在底物过量的条件下, 该重组酶的体外最适反应温度是50℃; 最适pH为5.5~6; Zn²⁺ 是酶促反应有效的激活剂。【结论】重组家蚕PLK与来源于家蚕组织的PLK具有相同的催化性质。     

转哺乳动物cyp2e1基因烟草植株再生及其分析

哺乳动物肝细胞中cyp2e1基因所编码的蛋白CYP2E1在代谢异型有机物方面起着重要作用,转cyp2e1基因植物可以代谢多种小分子有机污染物;但cyp2e1基因在植物体内的表达调控和代谢机理尚不完全清楚。文中将含有cyp2e1基因的质粒pSLD50-6和对照gus基因的质粒pKH200转入根癌农杆菌GV3101,利用根癌农杆菌转基因技术将cyp2e1基因和对照gus基因成功转入烟草,分别获得了转cyp2e1和gus基因再生植株。选取PCR鉴定的再生植株进行荧光定量PCR(qRT-PCR)分析,结果表明:在转录水平上,转cyp2e1基因烟草中,乙醇处理后cyp2e1基因的表达明显下降,苯和甲苯处理后cyp2e1基因的表达量稍有下降;而丙酮、甲醛处理和缺氧条件下cyp2e1基因的表达有不同程度的升高。此外,苯处理后,转cyp2e1基因烟草中NADPH-P450氧化还原酶和细胞色素b5酶的基因活性显著提高,说明烟草中NADPH-P450氧化还原酶和细胞色素b5酶与CYP2E1酶的解毒过程有关,可能起到哺乳动物体内的NADPH-P450氧化还原酶和细胞色素b5的功能,参与CYP2E1酶催化过程的电子传递链。     

Molecular cloning and expression of a novel CYP26 gene (cyp26d1) during zebrafish early development

Proper restriction of retinoid signaling by Cyp26s is essential for development of vertebrate embryos while inappropriate retinoid signaling can cause teratogenesis. Here, we report cloning and expression analysis of a novel cyp26 gene (cyp26d1) isolated from zebrafish. The predicted protein encoded by cyp26d1 consists of 554 amino acids. It exhibits 54% amino acid identity with human Cyp26C1, 50% with zebrafish Cyp26B1 and 38% with zebrafish Cyp26A1. Whole-mount in situ hybridization shows that cyp26d1 is first expressed in sphere stage, then disappears at 50% epiboly and resumes its expression at 75% epiboly. During segmentation period, cyp26d1 message is found at presumptive hindbrain. Double in situ hybridization with krox20 and cyp26d1 reveals that cyp26d1 is expressed in presumptive rhombomere 2-4 (r2-r4) at 2-somite stage. At 3-somite stage, cyp26d1 gene is expressed in r6 and pharyngeal arch (pa) one in addition to its expression at r2 and r4. At 6-somite stage, cyp26d1 message is present in continuous bands at r2-r6 and in pa1. This expression pattern is maintained from 10-somite stage through 21-somite stage except that the expression level is greatly reduced at r2 and r4. At 21-somite stage, cyp26d1 is also found in a group of cells in telencephalon and diencephalons. At 25-31h post-fertilization (hpf), the zebrafish cyp26d1 expression domain is extended to eyes, otic vesicles and midbrain in addition to its expression in hindbrain, telencephalon, diencephalons, and pharyngeal arches. At 35-48hpf, the expression of cyp26d1 is mainly restricted to otic vesicles, pharyngeal arches and pectoral fins and the expression level is greatly reduced.     

A cysteine protease gene is expressed early in resistant potato interactions with Phytophthora infestans

A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.     

Liver receptor homologue-1 (LRH-1) activates the promoter of brain aromatase (cyp19a2) in a teleost fish, the medaka, Oryzias latipes

The medaka, Oryzias latipes, like other fish, have two distinct aromatase genes, the ovarian (cyp19a1) and brain (cyp19a2) forms. We previously reported that Ad4BP/SF-1, a member of the NR5A subfamily, plays an important role in the regulation of cyp19a1 expression in medaka ovarian follicles during vitellogenesis. In the present study, we investigated whether liver receptor homologue-1 (LRH-1), another NR5A subfamily member, is involved in the regulation of cyp19a2 expression in the medaka brain. In situ hybridization analysis revealed that LRH-1 was expressed in the hypothalamus, where it colocalized with aromatase (cyp19a2). We then showed by transient transfection assays that LRH-1 was able to increase expression of a cyp19a2 reporter gene in various mammalian cell lines, and that mutation of a putative LRH-1 binding site within the cyp19a2 promoter abolished this effect. Taken together, these findings suggest that LRH-1 plays a role in regulating cyp19a2 expression in the medaka brain. This is the first to demonstrate in vitro the activation of brain aromatase by LRH-1 in the vertebrate brain.     

Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins.     

Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus

Fish have diverse sex determination and differentiation. DMRT1 and aromatase are conserved in the phyla and play pivotal roles in sex development. Gobiocypris rarus is a small fish used as a model in aquatic toxicology in China and has been used to study the effects of environmental endocrine disruptors on gene expression, but its sexual development remains elusive. Here, we report the full-length cDNA of G. rarus dmrt1 and its expression along with the expression of cyp19a1a and cyp19a1b, two genes encoding gonad and brain type aromatases, in adults and during ontogenesis. Both cyp19a1a and dmrt1 are expressed in the ovary and testis but show sexual dimorphism. Expression of cyp19a1a in the ovary is higher than in testes and dmrt1 follows the opposite pattern. Juvenile gonad histology changes at 15 days after hatching. The dimorphic expression of dmrt1 and cyp19a1a appears from 5 days after hatching, which is earlier than histological change. cyp19a1b is expressed coordinately with cyp19a1a until 15 days after hatching. These results show that dmrt1 and cyp19a1a play important roles in sex determination and sex differentiation in G. rarus.