【作者】 王勇；

【导师】 陈克平；

【作者基本信息】 江苏大学 ， 食品科学， 2008， 博士

【摘要】 bHLH(basic helix-loop-helix,碱性螺旋-环-螺旋)转录因子构成了真核生物蛋白质中的一个大家族,其成员在生物的生长发育调控过程中起着极为重要的作用。bHLH基序约含60个氨基酸,由一个能与DNA结合的碱性区域(basic region)和α螺旋1-环-α螺旋2(Helix 1-Loop-Helix 2)组成,其中环的长度在不同bHLH蛋白中会有差异。自从1989年第一个bHLH蛋白的结构被解析以来,迄今为止已经有近1000条bHLH序列在不同生物中得到鉴定。动物bHLH因其调控基因表达的功能不同而被分成45个家族;此外,根据它们所作用DNA元件和自身结构特点又被分成6个组。A组包含22个家族,主要调控神经细胞生成、肌细胞生成和中胚层形成;B组包含12个家族,主要调控细胞增殖与分化、固醇代谢与脂肪细胞形成以及葡萄糖响应基因的表达;C组包含7个家族,主要负责调控中线与气管发育、昼夜节律、激活环境毒素响应基因的转录;D组只有1个家族,它与A组bHLH蛋白形成无活性的异源二聚体;E组有2个家族,调控胚胎分节、体节形成与器官发生等;F组也只有1个家族,调控头部发育、嗅觉神经元生成等。迄今为止,生物基因组中的bHLH家族成员数得到鉴定的动物共有15种,其中,人类118个、小鼠102个、河豚84个、文昌鱼78个、星形海葵68个、帽贝63个、Capitella sp.Ⅰ64个、果蝇59个、水蚤57个、紫海胆50个、红粉甲虫50个、玻璃海鞘46个、线虫39个、水螅33个、海绵16个。家蚕是重要的鳞翅目模式昆虫,家蚕基因组测序工作在2003年10月即已完成,而对于其中有多少个bHLH家族成员还未有报道。本研究采用生物信息学方法开展“家蚕bHLH转录因子家族成员的鉴定及其基因的电子克隆”方面的探索,目的在于鉴定出家蚕中bHLH转录因子家族成员的数目,并初步弄清家蚕中编码bHLH转录因子的基因结构。主要结果如下:1、家蚕基因组中存在52个bHLH转录因子编码区域,这一结果与其它昆虫中的bHLH数目相近。其中,在12个家蚕bHLH基序的编码区域中有内含子,1个家蚕bHLH基序在基因组中存在两个为其编码的区域,分别位于Ctg038409和Ctg063456上。2、家蚕52个bHLH转录因子成员的分组及其家族归属情况为:A组21个,分别为BmASCa1、BmASCa2、BmASCa3、BmASCa4、BmMyoD、BmE12/E47、BmNgn、BmAtonal、BmMist、BmBeta3、BmNet、BmDeliah、BmMesp、BmTwist、BmParaxis、BmMyoRa、BmHand、BmPTFa、BmPTFb、BmSCL和BmNSCL;B组10个,分别为BmSRC、BmMyc、BmMnt、BmMax、BmUSF、BmMITF、BmSREBP、BmAP4、BmMLX和BmTF4;C组12个,分别为BmClock1、BmClock2、BmClock3、BmARNT、BmBmal1、BmBmal2、BmAHR1、BmAHR2、BmAHR3、BmSim、BmTrh和BmHIF;D组1个,即BmEmc;E组7个,分别为BmHey1、BmHey2、BmH/E(spl)1、BmH/E(spl)2、BmH/E(spl)3、BmH/E(spl)4和BmH/E(spl)5;F组1个,即BmCOE。在家蚕基因组中没有发现编码ASCb、NeuroD、Oligo、MyoRb、Figα和Mad家族bHLH蛋白的区域。3、应用电子克隆方法绘制出了11种家蚕bHLH的基因结构,分别是:BmASCa4、BmAtonal、BmBeta3、BmMesp、BmTwist、BmParaxis、BmMyoRa、BmMyc、BmUSF、BmEmc和BmH/E(spl)4;另外,对5种家蚕bHLH蛋白(BmMLX、BmTF4、BmClock1、BmARNT和BmHIF)的编码区域有了一定的了解。4、预测出了5种家蚕bHLH蛋白的功能,具体为:BmASCa4由l’sc基因编码,具有促进神经前体形成的功能;BmAtonal由αto基因编码,具有调控弦音器与光感受器形成的功能:BmTwist的作用是控制中胚层谱系分化以及抑制MyoD转录因子的功能;BmMyoRa的功能是抑制肌肉细胞的生成;BmEmc与其它bHLH蛋白形成无DNA结合能力的异型二聚体来对生长发育过程实施负调控。5、本研究还还对人类、大鼠、小鼠、红粉甲虫、果蝇、埃及伊蚊、非洲疟蚊、蜜蜂、鸡、蟾蜍、斑马鱼和海胆的emc基因做了电子克隆。从它们的结构上看,随着生物进化程度的提高,emc基因的外显子和内含子长度似乎有逐步缩短的趋势,倾向于支持内含子早现说。本研究所获得的结果为今后开展家蚕生长发育调控机理方面的研究积累了基础资料,也为进一步开展这些基因的表达谱分析、不同物种间bHLH基因结构与功能的比较等方面的探索打下了良好基础。更多还原

【Abstract】 Basic helix-loop-helix transcription factors form a super family of eukaryotic proteins.Its members play crucial roles in the regulation of organismal growth and development.The bHLH motif is composed of approximately 60 amino acids.It has one basic region,which is capable of binding DNA,and a HLH(Helix 1-Loop-Helix 2) structure,in which the loop region varies in different bHLH proteins.Since the discovery of the first bHLH protein in 1989,nearly 1000 bHLH sequences have been identified in various organisms.Animal bHLHs have been classified into 45 families according to their varied roles in regulating gene expression.Besides,they are divided into 6 groups based on their target DNA elements and their own structural characteristics.Group A has 22 families,They mainly regulate neurogenesis, myogenesis and mesoderm formation.Group B has 12 families.They mainly regulate cell proliferation and differentiation,sterol metabolism and adipocyte formation,and glucose-responsive gene expression.Group C has 7 families.They are responsible for the regulation of midline and tracheal development,circadian rhythms,and for the activation of gene transcription in response to environmental toxins.Group D has only 1 family.It forms inactive heterodimers with group A bHLH proteins.Group E has 2 families,which regulate embryonic segmentation,somitogenesis and organogenesis etc. Group F also has 1 family.It regulates head development and formation of olfactory sensory neurons etc.So far,the total number of animals of which bHLH family members have been identified is 15.The members identified in each species are 118 in human,102 in mouse, 84 in puffer fish,78 in lancelet,68 in sea anemone,63 in Lottia gigantea,64 in Capitella sp.I,59 in fruit fly,57 in Daphnia pulex,50 in sea urchin,50 in red flour beetle,46 in sea squirt,39 in nematode,33 in Hydra magnipapillata and 16 in sponge.Silkworm(Bombyx mori)is an important model insect of Lepidoptera.Silkworm genome sequencing project has been finished in October 2003.However,there has been no report on how many bHLH family members existing in the genome.This study employed bioinformatic methods to conduct studies on "Identification of bHLH transcription factor family members in Bombyx mori and in silico cloning of their genes".Its objectives includes to find out the number of bHLH transcription factor family members in silkworm and to explore gene structures that code for the bHLH transcription factors.The major results obtained are as follows:1.The silkworm genome has regions that code for 52 bHLH transcription factors. This figure is comparable with those in other insects.Twelve of the silkworm bHLH motifs have introns.One of them has two coding regions,locating on Ctg038409 and Ctg063456 respectively.2.All 52 identified silkworm bHLH transcription factors have been assigned into their corresponding groups and families.It was found that group A has 21 silkworm bHLH members.They are BmASCal,BmASCa2,BmASCa3,BmASCa4,BmMyoD, BmE12/E47,BmNgn,BmAtonal,BmMist,BmBeta3,BmNet,BmDeliah,BmMesp, BmTwist,BmParaxis,BmMyoRa,BmHand,BmPTFa,BmPTFb,BmSCL and BmNSCL.Group B has 10 members.They are BmSRC,BmMyc,BmMnt,BmMax, BmUSF,BmMITF,BmSREBP,BmAP4,BmMLX and BmTF4.Group C has 12 members.They are BmClock1,BmClock2,BmClock3,BmARNT,BmBma11, BmBma12,BmAHR1,BmAHR2,BmAHR3,BmSim,BmTrh and BmHIF.Group D has one member,BmEmc.Group E has 7 members.They are Hey1,BmHey2, BmH/E(spl)1,BmH/E(spl)2,BmH/E(spl)3,BmH/E(spl)4 and BmH/E(spl)5.Group F has one member,BmCOE.Coding regions for ASCb,NeuroD,Oligo,MyoRb,Figαand Mad families were not found in silkworm genome.3.Structural information of 11 silkworm bHLH genes was obtained through in silico cloning.They are BmASCa4,BmAtonal,BmBeta3,BmMesp,BmTwist, BmParaxis,BmMyoRa,BmMyc,BmUSF,BmEmc and BmH/E(spl)4.Partial structural information of 5 silkworm bHLH genes was also obtained.They are BmMLX,BmTF4, BmClock1,BmARNT and BmHIF.4.Functions of 5 BmbHLH proteins have been deduced,among which BmASCa4 is encoded by l’sc gene and promotes formation of neural precursers;BmAtonal is encoded by ato gene and controls the formation of chordotonal organs and photoreceptors;BmTwist plays roles in controlling differentiation of mesodermal lineage and inhibiting functions of transcription factor MyoD;BmMyoRa inhibits the formation of muscle cells;and BmEmc exerts negative control through forming heterodimers that lack DNA-binding ability with other bHLH proteins.5.Emc gene structures have also been explored in human,rat,mouse,red flour beetle,fruit fly,Aedes aegypti,Anopheles gambie str.PEST,honeybee,chicken, Xenopus,zebrafish and sea urchin.Based on their structures,it seems that,as the evolutionary level of the organisms heightens,exon and intron lengths of the Emc genes shorten gradually,tending to support the introns-early theory.The achievements of this study accrued fundamental knowledge for future studies on regulating mechanisms of silkworm growth and development.They can also facilitate further studies on expression patterns of the bHLH genes and on comparison of bHLH gene structures and functions between different species.更多还原