【作者】 黄健华；

【导师】 黄勇平；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 动物学， 2007， 博士

【摘要】 家蚕(Bombyx mori)是我国重要的经济昆虫,一生经历卵、幼虫、蛹和成虫四个发育时期,是完全变态型昆虫,它是研究昆虫变态发育的理想模式昆虫,也是鳞翅目昆虫的模式代表。研究家蚕生命过程中的各种基因及其表达特征是认识家蚕作为一种重要经济昆虫并对其进一步加以利用的重要前提。通过研究不但对昆虫变态发育研究有着重要影响,也能比较全面地认识家蚕生物学特性的分子调控机制,为提高蚕茧产量、改善蚕茧品质提供理论基础和技术支持,同时也有助于揭示更多鳞翅目昆虫的生命现象和本质。由于家蚕与鳞翅目昆虫高度同源性的特点,对家蚕的研究成果也可作为分子水平上认识重要鳞翅目害虫发生机理及寻求种类特异的防治靶点的数据来源。基因表达系列分析技术(Serial analysis of gene expression, SAGE)具有定量、高通量分析基因表达的特点,因此它能用于研究基因转录本在表达水平上发生的各种变化。它不仅能够提供简单生物个体或特殊类型的细胞和组织全基因表达图谱,寻找和证实一些差异表达基因;而且还可以利用SAGE方法获得新的基因标签,并进而筛选候选新基因;另外,标签序列对全基因组序列进行染色体基因注释也是SAGE研究的一个热点。本文开展了SAGE技术在家蚕功能基因研究中的应用,从以下四个方面入手并获得了相应的结果:第一,利用SAGE方法检测家蚕正常型和钴-60处理后辐射型胚胎之间的基因差异表达水平。同时构建了正常型和辐射型两个SAGE文库,分别得到了69100和57457个标签序列,其中特异标签序列分别为17718和15531个。对所构建的文库进行比较分析,总共发现了673个基因在表达水平上发生了显著的变化,它们必须同时符合P<0.01和相差3倍以上两个条件。其中292个基因经过辐射后,表达水平发生了下调,而381个基因经过辐射诱导后表达水平发生了上升。经过辐射后,细胞色素氧化酶,硫氧还原蛋白酶和一些核受体因子的基因表达水平受到了上调,而细胞骨架,表皮蛋白等相关基因的表达水平发生了下降,这些数据将为进一步了解家蚕胚胎发育和辐射诱变的分子机理提供了十分有效的证据。另外,通过GLGI(Generation of longer cDNA fragments for gene identification)技术,一方面验证了标签匹配的正确性,另一方面也能够获得大量的未知基因序列。通过对正常胚胎与辐射胚胎基因序列标签的分析也为进一步研究家蚕的基因表达特征建立了技术体系。第二,通过SAGE技术勾画了家蚕一个世代四个不同发育时期的基因表达谱,从而在转录本表达水平上,较为详尽的描述了家蚕的变态发育。经过分析这四个SAGE表达文库,总共得到了257964个SAGE标签,其中特异标签数目为39485个。依据标签检测到的次数分类,有14.1%的标签表达丰度≥5次,有24.2%的标签表达丰度在2-5次之间,而余下的61.7%为表达次数都为1次。这说明大量的转录本都处于一个低丰度表达的水平。经过和现有家蚕数据库中的基因序列比较分析,发现大约35%的SAGE标签能够匹配到已知基因序列。同时结果也显示,在不同的发育时期,有相当一部分基因在表达水平上发生了显著的差异,这类基因具有明显的时期表达特异性。SAGE标签序列结合GLGI技术,能够得到一批有重要意义的新基因序列,为下一步的功能研究奠定了基础。第三,对已经构建的家蚕四个不同发育时期的SAGE文库进行分析,发现有一批显著差异表达的标签,通过SGR的技术手段(综合了SAGE,GLGI和RACE三种方法)获得了相应的全长cDNA序列,其中一个标签序列TGTCGTCCTT,在蛹期具有很高的表达水平,它在卵、幼虫、蛹和成虫期的表达次数依次为:2,1,56和5,我们结合GLGI技术,得到了240个碱基长度的3’EST序列。为了进一步研究该基因的功能,依据3’EST序列设计引物,通过5’RACE的方法得到了该基因的全长cDNA序列。通过BLAST同源比较分析,该基因为家蚕鞣化激素Bursiconα亚基基因。接着利用实时定量荧光PCR技术深入研究了该基因在家蚕不同发育时期的基因表达水平,同时进行体外转录双链RNA,再将双链RNA显微注射到家蚕蛹并检查基因干涉RNAi后的效果,以便证实该基因的具体功能。结果表明,干涉后蛹期Bursicon基因的表达受到了显著影响,成虫期翅膀的伸展呈现非正常状态。进一步利用DD-RT-PCR方法进行研究,发现在Bursicon基因被干涉后,几个相关的基因表达丰度也相应地受到了不同程度的影响,其中包括一个和昆虫翅膀肌肉发育相关的海藻糖酶基因。以上的结果表明家蚕鞣化激素α亚基基因在成虫翅膀发育伸展过程中起到了极为重要的作用,而且这一作用可能是通过海藻糖酶基因参与调节而实现翅的鞣化。第四,2004年,中国科学家公布了6X基因组的家蚕全基因组框架图的测序结果,但是其测序结果还无法一一对应到相应的家蚕染色体上,同时也无法进行染色体的基因注释。在SAGE标签比对到的家蚕已知功能基因序列中,寻找CAPS分子标记位点,结合本课题组构建的SSR分子标记遗传连锁图,能够将基因定位到相应的染色体上。另外可将475个家蚕Scaffold序列精确定位到28条染色体上,然后结合获得的SAGE标签序列,对已经定位到染色体上的Scaffold序列进行了基因注释。总共注释基因1444个,其中特异基因909个,注释的基因组序列总长为15Mb,约为一个染色体的长度,同时也得到了这些功能基因在家蚕四个不同发育时期的表达丰度情况。更多还原

【Abstract】 The domesticated silkworm, Bombyx mori, is one of the most important economic insects in China, India, and many other developing countries owing to its propagation on a large scale and its utilization in silk production. Silkworm is one member of the holometabolous class of insects, which possesses four distinguished developmental stages, i.e., egg, larva, pupa, and adult. It is an ideal model for studying metamorphosis. Genetically, silkworm is one of the best-studied lepidopteran insects because large number of mutants has been identified. Lepidoptera also include highly destructive agricultural pests that cause widespread economic damage to crops and trees. Consequently, information on silkworm gene expression adds to our understanding of silkworm metamorphosis and provides novel candidate targets for pest control.Serial analysis of gene expression (SAGE) is one of the most versatile methods for genome-level studies of gene expression. The SAGE technology samples short sequence tags (14-bases long) from mRNA transcripts found in the population of interest. Each tag contains sequence information that can identify, in basic local alignment search tool (BLAST) analysis, the transcript from which an individual tag has been derived. The frequency of each tag in the SAGE library is an accurate estimate of the abundance of its corresponding mRNA transcript. Numerous groups have successfully used this technique and have described the SAGE protocol in detail. SAGE analysis has been used to study gene expression in a wide range of organisms, including yeast, Arabidopsis thaliana, rice, mouse, and human. SAGE is particularly well suited to the different gene expression analysis of organisms whose genomes are not completely sequenced, since it does not require a hybridization probe for each transcript and provides quantitative information on gene expression that is not readily obtainable using other methods. Also SAGE is suited to do chromosome annotation and find novel genes. Our researches including the following aspects:Firstly,SAGE was used to examine the profile of expressed genes during embryonic development in the domesticated silkworm, B. mori, after irradiation with cobalt-60. A comparison of the SAGE sequence tags derived from irradiated embryos with those from normal embryos revealed 673 differentially expressed genes (p < 0.001 and at least 3 folds change). Of these, 292 genes were highly expressed in normal embryos and 381 genes were highly expressed in irradiated embryos. These results provide valuable information for understanding the mechanisms of radiation-induced changes in gene expression. In addition, we found that the generation of longer cDNA fragments from SAGE tags is an efficient way to identify genes, thereby facilitating the analysis of large numbers of unknown genes.Secondly,we used SAGE to derive profiles of expressed genes during the developmental life cycle of the silkworm, and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool (BLAST) on the EST database, 35% of the tags matched known silkworm expressed sequence tags (ESTs). SAGE demonstrated that a number of the genes were up- or down- regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags (GLGI) constituted the most efficient method for gene identification, which facilitated the analysis of a large number of unknown genes.Thirdly,basing on the different expression SAGE tag: TGTCGTCCTT, we cloned the full-length cDNA sequence of silkworm bursicon gene and then studied the role of the gene function in wing expansion. Its gene expression at different developmental stages has been investigated in the silkworm, B. mori. Bursicon gene was expressed at low levels in larvae, high levels in pupae, and low levels again in adults. Then, we injected the double-stranded bursicon RNA into B. mori pupae to test RNA interference. The level of bursicon mRNA was reduced significantly in pupae, and a deficit in wing expansion was observed in adults. In addition, the differential display reverse transcription polymerase chain reaction (DD-RT-PCR) was used to reveal differences in the expression of transcripts in response to the inhibition of bursicon. In conclusion, bursicon plays a key role in the stereotyped behavioral program involved in wing expansion.Fourthly, in 2004, the draft genome (-428.7 Mb) of the domesticated silkworm, B. mori has been sequenced by the whole-genome shotgun method and should provide important insights into the functional genomic research. However, this genomic data was short of detailed gene annotation, even the chromosome location. Basing on the published SSR linkage map, 475 scaffolds have been located in the 28 chromosomes. Combined with the SAGE tags, these scaffolds also have been annotated. There were 1444 transcripts have been found in the totally 15 Mb genome and the number of unique gene is 909. From the data, we also get the gene expression level in the silkworm development stages about the located genes.更多还原