昆虫鞣化激素研究进展

鞣化激素是调控昆虫体壁黑化及翅伸展的一类激素, 是由BURS和PBURS两个亚基组成的一种异源二聚体蛋白质。BURS和PBURS亚基在结构及其进化上相对较为保守, 氨基酸序列中均含有11个半胱氨酸残基。鞣化激素主要是在胸腹神经节中合成的, 一旦释放到血淋巴就与其受体LGR2结合进而激活cAMP/PKA信号, 从而促进酪氨酸羟化酶(tyrosine hydroxylase, TH)的磷酸化。活化后的TH将酪氨酸(tyrosine)转变为多巴（DOPA）, 引起昆虫表皮鞣化。同时, cAMP/PKA信号也引起翅真皮细胞凋亡从而促进翅的伸展。除了鞣化激素异聚体调控表皮鞣化及翅的伸展外, BURS亚基或PBURS亚基组成的同源二聚体经IMD路径, 激活转录因子Relish调控昆虫的免疫反应。本文就鞣化激素分子结构特性、 作用机制及功能等方面的研究进展进行了综述, 旨在为进一步研究昆虫鞣化激素提供借鉴和参考。     

禾谷缢管蚜鞣化激素基因克隆及功能分析

【目的】探讨禾谷缢管蚜 Rhopalosiphum padi （Linnaeus）鞣化激素基因的发育表达模式及功能。【方法】采用转录组测序得到禾谷缢管蚜鞣化激素基因bursicon-α 和 bursicon-β cDNA序列。通过实时荧光定量PCR方法,分析该基因的发育表达模式。利用RNA干扰（RNA interference, RNAi）介导 bursicon-α 和bursicon-β 沉默,分析鞣化激素的功能。【结果】序列分析结果显示,禾谷缢管蚜鞣化激素α亚基基因（bursicon-α）cDNA序列开放阅读框为480 bp,编码159个氨基酸残基;β亚基基因（bursicon-β）cDNA序列开放阅读框为417 bp,编码138个氨基酸残基。时序表达分析表明,鞣化激素两个亚基基因在禾谷缢管蚜整个发育期均有表达,以1龄若蚜期表达量最高;成蚜有翅个体中表达量显著高于无翅个体。RNAi介导的 bursicon-α 和 bursicon-β 沉默均能显著抑制禾谷缢管蚜成蚜表皮的黑化。【结论】研究结果表明,鞣化激素在禾谷缢管蚜体壁黑化中发挥着重要作用。该结果为进一步研究鞣化激素在蚜虫生长发育过程中的生理功能提供了基础资料。     

小菜蛾鞣化激素基因克隆及其功能分析

鞣化激素是调节昆虫表皮骨化和翅膀发育的一种神经激素, 尽管已经在许多不同种昆虫上克隆了鞣化激素基因, 但是关于小菜蛾 Plutella xylostella鞣化激素及其基因的研究至今未见报道。本研究克隆了两个小菜蛾鞣化激素基因Pxbursα和Pxbursβ （GenBank 登录号分别为KF498645和KF498646）全长cDNA, 其序列长度分别为537 bp和360 bp, 与已报道的其他昆虫的鞣化激素氨基酸序列一致性分别为51%~68% 和37%~57%。实时定量PCR分析发现Pxbursα和Pxbursβ均在蛹期表达量高, 而在幼虫期和成虫期的表达量低。以Pxbursα部分序列的双链RNA（dsRNA）饲喂小菜蛾4龄末期幼虫, 发现蛹期Pxbursα的表达受到了显著抑制, 小菜蛾的发育停滞在蛹期而无法正常羽化, 并最终死亡。由此推测, 小菜蛾鞣化激素基因在蛹期的大量表达对其生长发育和羽化具有重要的作用。     

DNA甲基化通过调控细胞自噬影响家蚕翅的发育

[目的]本研究旨在探索DNA甲基化是否通过调控细胞自噬进而影响家蚕Bombyx mori翅的发育.[方法]分别用1和2 μg DNA甲基化特异性抑制剂5-aza-dC处理家蚕卵巢Bm12细胞和家蚕预蛹,荧光显微镜下观察Bm12细胞的数量,利用dot blot检测Bm12细胞中DNA甲基化水平,利用溶酶体染色检测细胞自噬...     

Hippo信号通路在乳腺癌中的调控机制及作用

Hippo信号通路是调控器官大小和肿瘤发生发展的关键通路,近年来受到广泛的关注。TAZ/YAP作为哺乳动物中Hippo信号通路两个核心下游效应分子,通过Hippo信号通路依赖性和非依赖性的机制受到细胞内外信号的严密调控。除了参与正常乳腺组织发育,Hippo信号通路还在人乳腺癌细胞的增殖、分化、凋亡、迁移、侵袭、上皮-间质转化和干性维持等多个过程中起着关键性作用。本文总结了Hippo信号通路的调控机制和调节信号,阐述了Hippo信号通路异常在乳腺癌发生发展中的作用,并讨论了其在乳腺癌中作为治疗靶点的临床策略。     

家蚕保幼激素结合蛋白Bmtol基因的克隆及功能分析

家蚕头部是一个神经中枢和感受的器官,其头部含有触角和感觉毛,感受外界的信号,并将外界信号传送到大脑进行反应。保幼激素主要是由咽侧体合成和分泌的,而保幼激素结合蛋白是保幼激素转运和发挥功能的载体,在昆虫体内具有极其重要的功能。文中通过Silk DB和NCBI数据库筛选并鉴定到一个新的具有保幼激素结合蛋白家族保守结构的蛋白Bm TOL,其编码基因编号为BGIBMGA003404(Gen Bank登录号:KY681053)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm TOL的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm TOL在头部都是高量表达,且Bmtol基因在起蚕时表达量较高,在5龄和蛹期表达量较低,而在化蛾后表达量又开始上调。免疫组化结果显示Bm TOL蛋白定位在头部的皮层、触角和脑中,推测其可能与头部信息传递有关,为家蚕的生长发育和行为调控提供重要的信息来源。     

昆虫翅多型的分子调控机制研究进展

生物如何适应复杂的环境变化是生物学研究的重要科学问题。其中,昆虫在长期的进化过程中形成了翅多型现象以适应复杂的环境,由此通过调控翅型的转换,对不同的环境条件做出响应,从而优化资源分配以平衡迁移或繁殖的需求,但目前对其分子调控机制仍不十分清楚。本文基于国内外最新研究进展,结合作者自己的研究,系统地综述了昆虫体内参与翅型转换调控的多种途径的研究进展,包括胰岛素信号通路、蜕皮激素信号通路、保幼激素信号通路、JNK信号通路、生物胺信号通路和病毒基因水平转移,指出了未来发展的方向。这些研究成果不仅对于进一步阐明昆虫翅多型的分子机制具有指导意义,也为开发以翅型调控为主要内容的害虫综合治理技术提供参考。     

中国米虾bursicon基因的功能及作用机制

为探讨米虾鞣化激素在其蜕皮周期及表皮角质层形成过程中的作用, 采用PCR技术克隆得到了米虾鞣化激素两个亚基基因的开放阅读框(ORF)序列。bursicon-α ORF全长441 bp, 共编码146个氨基酸; bursicon-β ORF全长411 bp, 共编码136个氨基酸。利用实时荧光定量PCR分析米虾整个蜕皮周期中鞣化激素2个亚基基因的表达特征, 结果发现, 鞣化激素bursicon-α和bursicon-β在米虾蜕皮周期的各个阶段的相对表达量存在差异, 在蜕皮前期(D期)相对表达量开始上升, 到D₃期时相对表达量最高, 蜕皮期E期相对表达量最低。RNA干扰(RNA interference, RNAi)介导bursicon-α和bursicon-β基因沉默后, 发现米虾的蜕皮周期延长, 表皮角质层明显变薄。结果提示, 鞣化激素(Bursicon)与新形成的外骨骼中角质层的加厚与硬化密切相关, 进而影响蜕皮时间。     

家蚕滞育的分子机理2.蛹期滞育激素基因的表达与滞育决定

滞育激素是由食道下神经节分泌的重要昆虫神经肽,诱导昆虫的滞育。选择具有ＲＮＡ聚合酶能够识别的启动子的质粒载体,将滞育激素ｃＤＮＡ克隆进去,在体外大量合成单一的滞育激素ｃＲＮＡ为参照,测定食道下神经节分泌滞育激素ｍＲＮＡ量来确定滞育激素的分泌量。结果证明食道下神经节分泌的滞育激素的数量决定昆虫的滞育。     

家蚕滞育激素－性信息素合成激活肽基因的表达

家蚕滞育激素－性信息素合成激活肽基因的表达徐卫华（中国农业科学院蚕业研究所,江苏镇江,２１２０００）山下兴亚（名古屋大学农学院,日本名古屋,４６４－０１）关键词滞育激素－性信息素合成激活肽基因;发育阶段;表达;家蚕昆虫是地球上最繁盛的物种,占地球上生...     

Effects of Diflubenzuron on the Development of Bombyx mori (Lepidoptera: Bombycidae)

ABSTRACT This study was conducted to determine the effects of diflubenzuron (DFB) on the growth and development of the silkworm, Bombyx mori (L.). The DFB treatment extended abnormally the larval duration and affected negatively on larval spinning of the 5th instar. All the larvae treated with high DFB concentration (2.5 × 10^-1 and 2.5x 10^-1μg/μl) lost their spinning capability and finally died, whereas 62% of larvae treated with low DFB concentration (2.5 × 10^-1μg/μl)) spinned cocoon. The larval weights depended sensitively on the DFB treatment period rather than on the DFB concentration. The DFB treatment decreased the larval maturity less than 6% without regard to the concentration and treatment period. All the larva, when treated with DFB before the 5th days of the 5th instar, were not matured.     

Reciprocal transplantation of wing discs between a wing deficient mutant (fl) and wild type of the silkworm, Bombyx mori

The wingless mutant flügellos ( fl ) of the silkworm lacks all four wings. Although wing discs of the fl seem to develop normally until the fourth larval instar, wing morphogenesis stops after the fourth larval ecdysis, probably caused by aberrant expression of an unidentified factor, referred to as fl . To characterize factor fl , the wing discs dissected from the wild-type (WT) and fl larvae were transplanted into other larvae and developmental changes of the discs were examined. When the wing disc from a WT larva was transplanted into another WT larva and allowed to grow until emergence, a small wing appeared that was covered with scales. Thus, the transplanted wing discs can develop autonomously, form scales and evert from adult skin. The WT wing discs transplanted into the fl larvae also developed at a high rate. However, the fl wing discs transplanted into the WT larvae did not develop during the larval to pupal developmental stages. These data suggest that the fl gene product (factor fl) works in the wing disc cells during wing morphogenesis. Its function cannot be complemented by hemolymph in the WT larva. It is also implied that the level of humoral factors and hormones required for wing morphogenesis are normally maintained in the fl larva.     

Rab14 from Bombyx mori (Lepidoptera: Bombycidae) shows ATPase activity

Rab GTPases are essential for vesicular transport, whereas adenosine triphosphate (ATP) is the most important and versatile of the activated carriers in the cell. But there are little reports to clarify the connection between ATP and Rab GTPases. A cDNA clone (Rab14) from Bombyx mori was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein bound to [³H]-GDP and [³⁵S]-GTPγS. Binding of [³⁵S]-GTPγS was inhibited by guanosine diphosphate (GDP), guanosine triphosphate (GTP) and ATP. Rab14 showed GTP- and ATP-hydrolysis activity. The Km value of Rab14 for ATP was lower than that for GTP. Human Rab14 also showed an ATPase activity. Furthermore, bound [³H]-GDP was exchanged efficiently with GTP and ATP. These results suggest that Rab14 is an ATPase as well as GTPase and gives Rab14 an exciting integrative function between cell metabolic status and membrane trafficking.     

家蚕Bcl-2家族基因 BmBuffy 的克隆及表达分析

【目的】本研究旨在获得家蚕 Bombyx mori Bcl-2家族同源基因,并分析其在不同组织和发育阶段的时空表达模式及功能。【方法】用cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆家蚕Bcl-2家族基因BmBuffy,利用SSR分子标记连锁分析确定其染色体定位。同时,用RT-PCR和qPCR技术分析该基因在家蚕幼虫及变态期间不同组织中的表达。【结果】克隆获得全长家蚕Bcl-2家族同源基因 BmBuffy,证明该基因位于第 4号染色体上,开放阅读框长879 bp,编码292 aa,预测其分子量大小为32.4 kDa,等电点为9.94,且第130-231位氨基酸之间存在1个Bcl-2_like Superfamily结构域。系统进化发育树表明,其与黑腹果蝇Drosophila melanogaster的DmBuffy关系最近,氨基酸序列一致性为27%。BmBuffy在幼虫组织中的表达结果显示,其在马氏管中表达量最高,而且在不同组织变态期的关键时间点均有明显变化。【结论】家蚕BmBuffy具有Bcl-2家族典型结构域,BmBuffy定位于第 4号染色体上,BmBuffy在家蚕变态期的组织生理变化中起到一定作用。本文为进一步研究家蚕Bcl-2家族基因的功能奠定基础。     

C-型咽侧体抑制素(AST-C)和促前胸腺激素释放激素(PTTH)基因在家蚕脑组织中的时空表达分析

【目的】咽侧体抑制素在昆虫体内具有重要的调控功能。本研究比较了家蚕Bombyx mori C-型咽侧体抑制素(allatostatin C,AST-C)与促前胸腺激素释放激素(prothoracicotropic hormone,PTTH)基因在不同发育阶段家蚕脑组织中转录表达的模式及其在脑组织的表达定位,以期为家蚕AST-C的功能研究提供重要的线索。【方法】利用脑组织芯片数据分析比较AST-C和PTTH基因在家蚕脑组织中的发育表达特征,RT-PCR验证其芯片数据,分析AST-C在不同发育阶段家蚕中枢神经系统中的表达模式,并用全组织包埋原位杂交技术对AST-C和PTTH在脑组织的表达进行定位。【结果】AST-C和PTTH在不同发育阶段家蚕脑组织中有相似的转录表达模式,且都在脑组织外侧一对神经分泌细胞中表达。【结论】AST-C可能与PTTH以相同的转录表达模式共同参与家蚕的变态发育调控。     

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.     

家蚕EGFR配体的鉴定和验证

表皮生长因子受体(EGFR)是广泛存在于后生动物中的多功能受体,其配体的种类、活化方式、配体与EGFR之间的相互作用以及激活的信号通路在哺乳动物中研究得较为深入。而非脊椎动物中,EGFR配体在各物种间差异较大,目前缺乏对除果蝇以外的其他昆虫EGFR配体的认识。通过同源比对、结构域预测、m RNA翻译起始序列分析和系统进化树构建,在家蚕中鉴定到2个EGFR的配体,命名为Bm EGF-1和Bm EGF-2。Bm EGF-1与果蝇Spitz有较高的同源性和一致的Rhomboid识别序列,Bm EGF-2为Vein的同源分子。经原核表达和纯化获得了Bm EGF-1胞外区段,利用Sf9细胞分泌Bm EGFR胞外区段,并通过pull-down实验验证了两者之间存在相互作用。在Bm E细胞中表达Bm EGF-1后,通过Western blotting检测到ERK和p38 MAPK的磷酸化水平增强,说明其不仅能激活经典的ERK信号通路,还可能通过p38 MAPK信号通路参与其他生理过程,为进一步研究EGFR配体在家蚕中的生物学功能提供了参考。     

结合SSR标记和STS标记对家蚕无鳞毛翅基因的定位

家蚕突变表型无鳞毛翅(non-lepis wing, nlw)由隐性基因nlw控制。由于家蚕雌性不发生交换, 文章采用有鳞毛翅品系P50和无鳞毛翅品系U06两个品系组配F1代及BC₁回交群体, (U06×P50)×U06和U06×(U06×P50)分别记作BC₁F和BC₁M, 根据已经构建的家蚕SSR分子标记连锁图谱及已经发表的有关序列对nlw基因进行了连锁及定位分析。得到8个与nlw基因连锁的SSR(Simple sequence repeat)标记和1个STS(Sequence-tagged sites)标记。BC₁F群中的所有正常翅个体均表现出与(U06×P50)F₁相同的杂合带型; 而所有无鳞毛个体带型与亲本U06一致, 为纯合型。利用BC₁M群体构建了关于nlw基因的遗传连锁图, 连锁图的遗传距离为125.7 cM, 与nlw基因最近的引物为STS标记cash2p, 图距为11.4 cM。     

Cytopathological process by multiple nucleopolyhedrovirus in the testis of Bombyx mori L., 1758 (Lepidoptera: Bombycidae)

A cytopathological methodology was used to analyze infection by Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), a geographic isolate of the family Baculoviridae, in the caterpillar testes of the B. mori. Japanese B. mori strain caterpillar, fifth instar, were inoculated with BmMNPV and their testes were collected and processed for light and transmission electronic microscopy. Epithelial coating cells and interfollicular septa in testes were susceptible to BmMNPV. The first evidence of infection was detected on the 6th day post-inoculation (p.i.) in the external epithelium, and on the 7th day p.i. in the internal epithelium and interfollicular septa. Cytopathological characteristics consisted of hypertrophied nuclei, the formation of virogenic stroma, and the occlusion of virions in polyhedron protein crystals in several stages of development. At the end of the infectious process, cell lysis and release of polyhedra into the extracellular medium occurred. Histopathology revealed early infection foci in the surrounding regions of tracheal insertions, thus underlining the role of the trachea as an infection-spreading organ in insects. This spreading occurs through penetration of the basal lamina, which facilitates entry of the budded virus into the testis. Additionally, an alignment of a partial sequence of the ORF 14 of the BmMNPV geographic isolate with other NPV certified the virus genera.