| 其他摘要 | 家蚕(Bombyx mori)属于鳞翅目蚕蛾科，是重要的遗传实验动物。由于其丰富的遗传资源、实验操作的便利性，家蚕作为早期遗传学研究的著名材料已经被使用了愈百年，为早期的经典遗传学的发展做出了重要贡献。而且家蚕是人类唯一完全驯化的昆虫，驯化历史超过5000年，是研究人工选择机制的重要模式动物。家蚕不仅是重要的科学研究对象，也是著名的经济昆虫。蚕丝业是我国重要的传统产业和悠久中华文明的重要代表，为我国的经济和社会发展做出了巨大贡献，也是目前在国际市场上处于绝对优势的特色产业。所以对家蚕的研究非常重要。而在家蚕的众多性状中，产丝能力不仅是重要的驯化性状，具有重要的科学研究价值，也具有非常重要的经济价值。一方面，家蚕经过长期的人工选择和驯化，产丝能力和其祖先中国野桑蚕相比发生了非常显著的变化，对产丝能力变化背后遗传机制的研究将为研究人工选择的机制提供证据。另一方面产丝能力直接关系到蚕丝产量和质量，是蚕丝业育种过程中关注的重点性状。近年来随着蚕业的多元化及功能基因组学、材料科学等众多学科的快速发展，蚕丝制品及蚕丝蛋白也开始应用于生物化工、医疗、安保等等领域，应用前景非常广阔。然而局限于常规家蚕蚕丝蛋白的机械及生化性质，蚕丝在这些领域的广泛应用还需要更多的研究予以支撑。基于以上原因，本论文试图解析家蚕产丝能力进化的遗传学机制，并对蚕丝丝质性状改良进行了一些前期技术和基因的探索。首先，我们分析了家蚕改良品种、早期驯化品种和祖先野生种的丝腺转录组间的差异。家蚕在驯化和改良过程中，产丝能力经历了不同程度的改变。早期驯化种的产丝能力和野蚕相当。但是在被改良后的品种中，家蚕丝腺体积大幅增加，而且与之相关产丝能力加强，蚕茧产量大幅提高。我们通过全基因表达多样性分析、差异表达基因分析和共表达网络分析，对这一变化过程做了初步的研究和探讨。我们发现了大量富集于“translation”、“gene expression”、“ribonucleoprotein”、“macrosmolecular complex”“enzyme regulator”、“transmembrane transporter”、“macromolecule metabolic process”功能类别的基因。也发现了在家蚕中受到正选择的CCG012578.1（FAM46C）基因在改良品种中的表达量成倍数增加。这个基因在果蝇唾液腺中表达量也非常高，表明这个基因可能在丝腺中起到了重要的功能。其次我们通过转录组测序对家蚕中杂种优势现象进行了研究。杂种优势是家蚕育种的重要手段，目前生产用的品种基本都是蚕丝产量很高的杂交品种。我们的研究发现亲本和杂交后代差异表达基因中86.75%呈现的是非加性表达，而且52.26%的非加性基因呈现出来超亲表达模式。我们发现超高亲表达的基因会在“transmembrane transport”“signaling process”相关共能上富集。而超低亲表达的基因功能分析中，我们发现富集在DNA复制和细胞分裂相关的功能上，如“base excision repair”, “cell cycle”等。我们还发现三羧酸循环中10个关键性酶有七个超低亲表达，两个偏低亲表达，一个是保守表达，预示着杂交后代丝腺中能量代谢水平降低。这些研究为提高蚕丝产量提供了更丰富的遗传资源和手段。最后蚕丝丝质性状改良方面，我们计划将外源优质丝素基因转入到家蚕中来提高蚕丝品质。一方面，我们建立基于TALEN、CRISPR/Cas9 和piggyBac 转座子的高效转基因平台。通过TALEN和CRISPR/Cas9， 我们敲出了家蚕內源的丝素重链基因，以利于提高外源蛋白的表达量和方便提纯。同时我们也构建家蚕丝素重链外源蛋白表达载体。另一方面，我们尝试挖掘外源物种优质的丝素蛋白基因，期望通过转基因平台在家蚕里高效表达这些优秀的基因，产生高质量的天然丝。我们通过对三种结网蜘蛛、六种野生绢丝昆虫的转录组分析，对蜘蛛丝相关基因和大蚕蛾科丝素蛋白相关基因的结构和性质进行了研究。并且转录组数据为我们获得这些基因的全长提供了参考。; Bombyx mori is a kind of lepidoptera bombycidae insects. It is an important genetic model animal. Because of its numerous genetic resources and the convenience of experimental operation, silkworm has become a famous study object for a hundred years and make lots of contributions to the development of genetics. Besides, silkworm is the only domesticated insect, which undergo more than 5000 years of domestication. It is an important mode animal for studying artificial selection. In addition to its roles in scientific studies, silkworm is also a famous economically important insect. Sericulture represents an important traditional industry and traditional culture in China and has made unignorable contributions to the development of economy and society in China. So, the study of silkworm is very important. Specifically, among the characters of silkworm, the study of silk production ability is necessary, not only because it is a significant domesticated phenotype, but also it closely connects with economic outcomes. Nowadays, silk has broad applications in biochemical engineering, medical care, security and so on. However, the poor mechanical and biochemical properties of silk have impeded its application. Considering all the above situations, we are trying to interpret the genetic mechanisms of silk production ability and make preliminary efforts in technology and useful genes for promoting silk properties. .Firstly, we compare the transcriptomes of improved silkworm strains, early domesticated silkworm strains and Bombyx mandarina. The silk production ability has experienced promotion to different extent in domestication or improvement. The cocoons of early domesticated silkworm strains are nearly same as Bombyx mandarina, while the improved silkworm strains have much bigger cocoons. We investigate this phenotype by analysis of genome-wide gene diversity, differential expressed genes and co-expression gene network. We found that hundreds of genes are enriched in “translation”, “gene expression”, “ribonucleoprotein”, “macromolecular complex”, “enzyme regulator”, “transmembrane transporter”, “macromolecule metabolic process” GO categories. We also found that a positively selected gene CCG012578.1（FAM46C）had up-regulated expressions in improved silkworm breeds. The homolog of this gene has the highest expression levels in salivary glands of Drosophila, which indicate the import roles of this gene in silk gland.Secondly, we studied the heterosis vigor phenomenon of silk production ability by using transcriptome and genome sequencing data. Heterosis is the most important approach for silkworm breeding. In our study, we found that among the differential expressed genes between parents and F1, 86.75% genes present non-additive expression mode. Specially, 52.26% non-additive expressed genes are transgressively expressed. We found the transgressively up regulated genes are enriched in “transmembrane transport”, “signaling process” related GO categories, while the transgressively down regulated genes are enriched in cell replication and cell division related categories, like “base excision repair”, “cell cycle”. Importantly, we found among the ten key enzymes involved in TCA cycle, seven has transgressively down regulated expressions, two has dominantly down regulated expressions and one has conserved expression. Our studies could advance the research of heterosis and supply more genetic resources and approaches to improve silk yield.Finally, we made preliminary efforts to improve the properties of silk by ectopic expression of genes which produce high quality silk, like spider silk. On one hand, we established TALEN, CRISPR/Cas9 and piggyBac technologies in silkworm. By TALEN and CRISPR/Cas9 systems, we successfully knock out the endogenous fibroin heavy chain gene in silkworm, which could help improve the productions of exogenous proteins. On the other hand, we analysis the transcriptomes of three web spiders and six Saturniidae moths. We studied the structure and properties of spider silk genes and fibroin genes in Saturniidae species. In addition, the transcriptome date would provide the information for obtaining the full length of those genes. |
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