【作者】 童晓玲；

【导师】 鲁成；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2008， 博士

【摘要】 昆虫是地球上最早在空中飞行的动物。也是目前地球上种类最多、分布最广的一类生物。翅的发生使昆虫在觅食、寻偶、扩大分布和避敌等多方面获得了优越的竞争能力,是昆虫纲成为最繁荣的生物类群的重要原因。翅的特征也是研究分类和演化的重要依据。随着分子生物学的发展,迄今已取得了大量关于昆虫翅发育的研究成果,并表明翅发育的基本机制在昆虫间是比较保守的。但在漫长的进化历程中,主要由于自然选择的结果,昆虫翅的形态和功能出现了许多不同的类型,这些形态上的差异究竟源于哪些遗传信息的改变?其中的分子调控机制又是怎样的?这些问题需要深入研究。家蚕是鳞翅目昆虫的典型模式,也是目前完成基因组测序的唯一一种鳞翅目昆虫。家蚕翅发育相关基因的研究,对昆虫生理学、发育生物学以及生物进化等都具有重要的价值。家蚕在长期的人工选择压力下,已丧失了飞行能力,翅上斑纹严重退化,通过研究参与家蚕翅发育的基因,为揭示家蚕翅功能整体退化的分子机理奠定重要理论基础,对鳞翅目害虫的生物防治有重要意义。本研究借鉴模式生物果蝇关于翅的研究成果,利用家蚕的全基因组序列、ESTs以及基因芯片数据,对家蚕翅发育相关基因进行了生物信息学分析,同时采用克隆、RT-PCR、原位杂交和RNA干涉等技术对部分基因的表达以及功能进行了研究。获得的主要结果如下:1.家蚕翅模式决定基因的生物信息学分析前后轴和背腹轴两个级联调控途径对果蝇翅模式形成起着非常重要的作用,其中主要包括22个基因。通过同源检索,我们发现除vestigial(vg)基因外,其余21个基因在家蚕基因组中均存在同源基因。这些决定翅模式形成的基因大多在蝴蝶中也都有报道。这一结果表明,鳞翅目和双翅目昆虫翅模式决定的级联调控途径比较保守。分析家蚕和果蝇翅模式决定基因的结构和功能域,结果表明相应基因功能域序列相似性很高,进一步说明它们在翅发育中扮演着较为相似的角色。同时。部分基因(如Ubx,Dll等)在功能域外也表现目(如双翅目)的特异性。推测是由于昆虫在进化过程中翅的形态发生分化,相应基因的功能及其调控方式略显差异,使得在进化中其基因结构渐渐分化而表现出各自种群的特征。2.家蚕翅的形态发生与基因表达模式研究以N4为实验材料,观察家蚕5龄幼虫到化蛾前各时期翅原基及蛹翅(翅芽)的形态特征,表明5龄幼虫翅原基生长缓慢;蚕老熟后翅原基则迅速生长,其上的气管明显增多:化蛹前(W2末期),翅原基已经展开,与蛹初期翅芽大小相当。化蛹后,蛹的翅芽包被于表皮之下,整个蛹期,翅芽的大小和形状都没有太明显的变化,只是翅芽发育逐步完善的过程,包括翅脉的硬化、鳞片的形成、花纹的出现等。在化蛹后第2d(P2)始见翅脉逐步形成,第3d(P3)可见翅芽的边缘出现初生的鳞毛,第6d(P6)鳞毛和翅脉基本形成。同时,对5龄幼虫的翅原基进行石蜡组织切片,调查了在发育过程中翅原基内部结构的变化。基于对家蚕翅模式决定基因的生物信息学分析结果,调查这些基因在翅发育过程中的表达模式。结果显示决定翅模式的基因在家蚕翅发育过程中都有大量表达。幼虫期,多数基因的表达量都呈逐渐升高的趋势;化蛹后,其表达量逐渐降低;羽化前,几乎所有这些基因都不再表达。从整体上看,这些基因的表达高峰主要集中在幼虫末期到化蛹初期,如wg,hh,srf,fng等。形态观察的结果表明这段时期正是翅原基形态发生巨大变化的时候,推测这些基因的高表达与翅原基的形态变化有关。同时部分基因在化蛹后仍维持较高水平的表达,如inv,fng,cut,wg等,推测可能是因为这些基因除了决定翅的模式外还参与后期翅的形态特化,如翅脉、鳞毛和斑纹的发育等。3.翅叶发育重要基因Bmwnt-1的研究参考人、果蝇、海葵的38个Wnt蛋白序列,通过序列比对,在家蚕9倍基因组中,找到9个同源基因,基于序列相似性,将其命名为:Bmwnt1,Bmwnt4A,Bmwnt5B,Bmwnt6,Bmwnt7,Bmwnt10A,Bmwnt10B,Bmwnt11和Bmwnt11A。在不同物种中,Wnt基因的数量不同。人的基因组有19个Wnt基因、低等的海葵有12个。而在昆虫(家蚕、果蝇和赤拟谷盗)中,Wnt基因的数量偏少,推测Wnt基因在进化过程中可能有基因丢失的现象,尤其在昆虫中。但Wnt的基因结构在进化过程中是比较保守的,常以成串的方式排列。家蚕有2个Wnt基因簇,Wnt11-Wnt1-Wnt6-Wnt10和Wnt7-Wnt5B-Wnt4,分别位于4号和28号染色体上。家蚕的Bmwnt-1基因是Wnt1亚型的一个同源体。分析双翅目和鳞翅目昆虫的Wnt1蛋白的分子结构,发现尽管Wnt-1蛋白在不同物种内都有较保守的结构域,但在进化过程中该结构域也在不断地分化,在目间表现山明显的差异性。对Bmwnt-1基因在翅发育过程中的表达谱分析表明,Bmwnt-1在5龄幼虫的翅原基中的表达量较高,一直持续到上簇后第2d(W2),化蛹后,Bmwnt-1的表达量逐渐降低。用Bmwnt-1dsRNA沉默该基因在翅原基中的表达,出现翅叶部分缺失,甚至完全消失的突变表型,不能很好的形成翅叶。由此说明家蚕的Bmwnt-1控制翅叶的发育。4.家蚕AS-C complex的研究鉴定了家蚕AS-C复合物,包括BmASH、BmASH2、BmASH3和Bmase 4个基因。通过序列的同源性分析得出家蚕BmASH、BmASH2、BmASH3是原神经基因,Bmase是神经前体基因。进化分析表明,AS-C complex的祖先最初就分化为原神经基因和神经前体基因。在大多数的昆虫中都只有这2个AS-C基因。但双翅目昆虫中,AS-C complex经历基因重复后分化出了4个基因,本研究发现家蚕有4个AS-C基因,是除双翅目以外的首例发现。不同物种中,尽管AS-C基因数目不同,但AS-C complex及其两侧基因的结构是非常保守的,遵循着一定的排列方式,即原神经基因位于神经前体基因的上游,在AS-C complex的两侧紧挨着一个yellow基因和一个细胞色素基因cyt P450。用半定量RT-PCR调查了4个AS-C基因在家蚕不同组织中的表达情况,发现其在翅原基中的表达量明显高于其他组织。进一步检测它们在翅发育过程中的表达,结果表明这4个AS-C基因都有较高的表达水平,其中BmASH2和BmASH3基因的表达模式非常相似,呈现共表达的趋势。在蝴蝶里,鳞毛前体细胞的第一次细胞分裂发生在化蛹后的24h左右,bASH1基因(蝴蝶AS-C的同源基因)在此时大量表达。在家蚕中,对应的时期里高表达的是BmASH基因,推测家蚕的BmASH基因与鳞毛的分化关。我们用RNAi研究BmAsh基因的功能,BmASH基因干涉后出现翅表面部分区域鳞片缺失的现象,且该区域毛孔也极少。表明BmASH基因在家蚕翅鳞毛的形成过程中起着至关重要的作用。5.BmSRF基因的功能研究克隆家蚕的血清应答因子BmSRF基因。BmSRF基因在翅发育过程的表达模式分析和翅原基中的原位杂交结果都表明,该基因参与翅的发育,主要的作用时期是化蛹后1、2d(P1、P2)。说明BmSRF基因对翅的形态特化具有非常重要的作用。我们用BmSRF dsRNA沉默该基因在翅原基中的表达,出现了翅叶上有囊泡和翅脉增多这两种突变表型,同时还伴有鳞毛增多的现象。初步认为BmSRF对翅的上下表皮的粘合和脉络的形成有重要作用。BmSRF基因干涉后出现了囊泡型翅的突变表型。该表型与家蚕突变螯虾蛹(cf)的表型较为相似,这引起了我们探究家蚕螯虾蛹突变形成原因的极大兴趣。通过对螯虾蛹和正常型翅原基的解剖,我们观察到在上簇后第1d(W1),突变型cf的翅原基中的气管的数量明显比野生型多,且较野生型显得凌乱,生出一些微气管。进一步对该时期二者的翅原基进行切片,观察发现在W1期野生型翅原基中气管发生迁移,而突变体cf的翅原基中其气管在迁移过程中极度拉长,并伴有气管融合的现象。鉴于此,我们用定量PCR检测了BmSRF基因在突变型cf和野生型翅发育过程中的表达情况。结果显示在突变型cf里BmSRF基因的表达水平比野生型高,尤其在上簇后第1d(W1)。这与我们对二者翅原基的形态观察结果一致,说明W1期是突变体cf形成的重要时期,推测BmSRF基因在该时期的过量表达或许是导致突变体cf形成的一个重要原因。更多还原

【Abstract】 Insects are the earliest species which could fly over the world, also are the most prosperous organism on the earth with wide distribution at present. The wings of insects make them taking many advantages in predation, mate choice, expanding distribution and safe-keeping, and so on. This is the most important reason for them to become the most prosperous organism. The characteristic of wings is the important basis for the researches of classify and evolution. Following the development of molecular biology, a great amount of outcomes about the development of insect wing had been obtained. It indicated that the basic mechanism in wing growth is quite conserved among different insects. However, the shapes and functions of insect wings have presented many different types after a long evolution course, mainly as a result of natural selection. Which genetic information changed during the evolution? Their molecular regulation mechanism is still unknown. More deeply researches will be required to answer these questions.Silkworm is the typical model insects in Lepidoptera, is also the only one lepidopterous insect which has been completed the genome sequencing at present. The study of wing development genes in the silkworm is significance for insect physiology, developmental biology, evolution, and so on. B. mori has taken place many notably changes during more than 5,000 years artificial breeding, for example, silkworm can not fly, and only some traces for eyespots and bands can be found on the corresponding position of wing. So, the wing of silkworm is really a rare model for the study of degerated wing patterns. Therefore, study on the regulative mechanism of silkworm wing development is important for prevention of Lepidoptera pests.Learning from advanced achievements about wing development in Drosophila, we performed a homology search on wing pattern genes in the silkworm genome by bioinformatics analysis based on the genome sequences, ESTs and the gene chip data. The spatio-temporal expression profiles and functions of several homologous genes have been analyzed by gene clone, RT-PCR, in situ hybridization and RNAi techniques. The major findings are as follows:1. Bioinformatics Analysis of Silkworm Wing Pattern Genes Antero-posterior axis and dorsal-ventral axis are the mainly two gene-regulatory hierarchies for the wing pattern determination in Drosophila, mainly includes 22 genes. Through the homology search, we found 21 genes have the homologous in the Bombyx mori genome except vestigial （vg）. Most of them also have been reported in the butterfly. It indicated that the gene-regulatory hierarchy in wing pattern is quite conserved between Lepidoptera and the Diptera.The gene sequence analysis indicated that the similarities of functional domain of corresponding wing pattern genes are very high between Bombyx mori and Drosophila. It further proved that they are playing more similar role in the wing development. However, partial genes （such as Ubx, Dll and so on） also display some differences outside the functional domain. We presumed that the differences of gene structures may be resulted from the differences of gene functions and the regulative ways to adapt for the environment during in the evolution process.2. The morphogenesis and gene expression pattern of silkworm wingWe investigated the development of the wing discs in size and form during 5^th instar larvae and pupal priod of N4. We found that the wing disc growth was slow in the prophase of the larva, while reach a peak just as wandering, and the number of trachea in the disc increased obviously. The wing disc spread out in the end of W2 day, the size of the wing bud nearly quite with that of in pupa. We also found that the pupal stage was a perfecting period of the wing buds, including the formation of vein and scales, with a little change of the size. We found the firstborn veins present on 2d pupa, the firstborn scales present on 3d pupa. The veins and scales were completely formed on 6d pupa. While, we performed the paraffin wax tissue slice on the wing discs of 5^th larva and investigated the changes of internal structure during wing growth.Based on the results of biological information analysis of the wing pattern genes, we investigated the expression profiles of these genes during in wing growth. The results showed that wing pattern genes expressed massively in wing. The expression of most genes showed increasing gradually in larval stage and reducing after pupated, nearly could not be detected in moth. In a word, the expression peaks of these genes mainly present in the stage from the end of larva stage to the initial of pupation, such as, wg, hh, srf,fng and so on. The results of morphous observation indicated that it was just the period during which wing disc taken placed immense changes in morphous. We presumed that the high expressions of these genes were related with the changes of morphous. In addition, some genes were also showed high expression after pupate, such as inv,fng, cut, wg, and so on. Maybe they were also required in the perfecting period of the wing buds, as formation of veins and scales.3. The research of Bmwnt-1 in wing blade developmentRefers to the 38 Wnt protein sequences of human, Drosophila, sea anemone, we found 9 Wnt genes in the Bombyx mori genome through the homology search. Based on sequence similarity, they were named as Bmwnt1,Bmwnt4A,Bmwnt5B,Bmwnt6,Bmwnt7,Bmwnt10A, Bmwnt10B, Bmwnt11 and Bmwnt11A. The numbers of Wnt genes are different in various species. There are 19 Wnt genes in human and 12 Wnt genes in sea anemone. However, there are fewer Wnt genes in insects, such as Bombyx mori, Drosophila and Tribolium castaneum. We presumed that the Wnt genes might lost in the evolution, especially in insect. However, the genome structures of Wnt genes were quite conserved. They are organized in tandem arrays in the genome. The Bombyx mori has 2 Wnt gene bunches, Wnt11-Wnt1-Wnt6-Wnt10 and Wnt7- Wnt5B- Wnt4. They were located at 4 and 28 chromosomes, respectively.The Bmwnt-1 gene belongs to the Wnt1 subtype. The analysis of protein structures of Wnt1 genes in Diptera and the Lepidoptera showed that they have conservative structural domains in the different species, but the gene structures also showed some specificity between Diptera and the Lepidoptera.In this study, we examined the expression profile of the Bmwnt-1 gene during wing development in the silkworm by semi-quantitative RT-PCR. The results showed that it expressed at high abundance in the wing disc from 5^th instar larvae to W2, and reduced gradually after pupate. We used RNAi to reduce the expression of Bmwnt-1 in wing disc and then assayed Bmwnt-1 expression and examined morph after injection. We observed the correlation between reduce of Bmwnt-1 expression and the partly absent of wing blade. It indicated that Bmwnt-1 play important role in the wing blade formation in silkworm.4. The research of AS-C complex in silkwormWe identified four AS-C homologue genes in the silkworm, proneural genes BmASH, BmASH2, BmASH3, and neural precursor gene Bmase.Phylogenetic analysis showed that the ancestral AS-C gene has independently divided into proneural-like and ase-like functions in the insect groups. Most of insects only have a proneural gene and a neural precursor gene. However, there are four AS-C genes in most of Dipteral insects resulted form gene duplication. In our study, we found that the silkworm also has four AS-C genes. It was the first found of another insect besides Diptera which present four AS-C genes. Though the numbers of AS-C genes are different in various species, the AS-C complex and its neighboring genes showed a conserved syntenic structure that the neural precursor gene lied downstream of the proneural genes, a yellow gene and a cyt P450 reside at two ends of the AS-C complex, the yellow gene lies upstream of the complex and the cyt P450 resides the downstream in most insects.To investigate the spatial expression patterns of the AS-C genes, we used semi-quantitative RT-PCR to analyze total RNA samples of 12 silkworm tissues on the sixth day of the 5^th instar larva. The results indicated that the expression levels of the four silkworm AS-C genes were obviously higher in wing disc than in other tissues. So we examined the expression profiles of the four AS-C genes during wing development, the results indicated that the four AS-C genes were all expressed at high abundance in wing disc, but their expression profiles were slightly different. The expression profiles of BmASH2 and BmASH3 were nearly the same. In butterfly, the expression of JcASH1 （AS-C homolog in the butterfly） was detected the following 24 hours after pupate, the critical period when the first scale precursors are formed. In silkworm, the peak level of the BmASH appeared at the onset of pupation, which was the time of the first scale precursors formed in butterfly. These results strongly suggested that the expression of BmASH in wing discs was mostly correlated with the development of the scales in silkworm. We used RNAi to reduce the expression of BmASH in wing disc at the late stage of the larvae and then assayed scale precursor formation molecularly by BmASH expression after injection and by morphological examination. We observed the correlation between reduce of BmASH expression and the absence of scales. It demonstrated that BmASH was essential for the scales differentiation and formation in silkworm.5. The function study of BmSRFgeneWe cloned the serum response factor （BmSRF） in silkworm. The results of expression profile and in situ hybridization of BmSRF gene in wing disc indicated that it play important role in the wing development. The expression of BmSRF gene reached the peak at P1, P2. We used dsRNA to reduce the expression of BmSRF in wing disc at the stage of the larvae. The expression of BmSRF after injection was nearly half of that in controls. And we observed the mutants defects range from wings with ectopic veins and intervein blisters to completely ballooned wings where the distinction between vein and intervein is lost, and there were more scales in the mutants. It demonstrated that BmSRF was essential for the formation of veins and interveins and the apposition of dorsal and ventral wing epithelia in silkworm.After silence of BmSRF gene, the silkworm presented the mutant phenotype that failure of apposition of dorsal and ventral wing epithelia. This phenotype was similar with that of cf mutant in silkworm. It caught our attentions to find the reason of the formation of mutant phenotype in cf. we dissected and observed the wing discs in cf mutant and normal. We found that there were more tracheas in cf than normal, and they appeared more disorderly. Furthermore, we found that the tracheas elongated and fused when the migration on the W1 stage in cf mutant by sliced, thus, the tracheas in the normal were just migrating on the same time. In view of this, we used real-time PCR to examine the expressions of BmSRF gene in mutant cf and wild type during wing development. The result showed that the expression level of BmSRF gene in cf mutant is higher than that in wild type, especially on the first day of wandering （W1）. It was consistent with the results of phenotype examination in the two materials. All of that indicated that the W1 stage is the most important period in which formed cf mutant, and the excessive expression of BmSRF gene perhaps was one of the most important reasons caused the cf mutant.更多还原