黑腹果蝇的Y染色体占雄性果蝇基因组的13%，含有约40Mb的DNA，对雄性果蝇的生育能力具有重要作用。尽管果蝇的Y染色体相当大，但其蛋白质编码序列的密度极低。果蝇基因组约有16000个基因，但仅有16个基因位于Y染色体上。Y染色体是由高度重复的DNA序列以及卫星DNA组成，几乎完全是异染色质。同时，Y染色体中的大部分序列未知，由此阻碍了我们对Y染色体上多数基因的功能研究。目前，我们对Y染色体上的基因间区是否存在功能性元件以及是否所有的Y染色体基因都为雄性果蝇生育所必需尚不清楚。 为此，我们利用双组分的CRISPR/Cas9系统和RNAi技术来逐个敲除或敲低Y染色体编码基因。除了先前已被证明基因kl-3导致雄蝇不育的作用外，我们发现两个假定的育性基因kl-2和kl-5同样对雄蝇育性具有重要的作用。此外，我们还发现另一个基因CCY也在雄性生育中发挥作用。本文中的研究表明， CCY、kl-2、kl-3和kl-5四个基因中的任何一个基因发生突变或RNAi敲低都会影响精细胞核的延伸过程，进而导致精细胞的个体化过程产生缺陷，包括延伸复合体（IC）和同步化的缺陷。本论文系统性地鉴定了Y染色体基因在雄性生育中的功能，发现了与育性相关的四个基因，并表明并非所有的Y染色体基因都为雄性果蝇生育所必需。 为进一步探究Y染色体功能，例如，Y染色体基因间区是否存在功能元件，以及Y染色体育性基因是否足够维持雄性果蝇的生育能力，我们应用合成生物学方法人工合成一条Y染色体进行研究。通过Golden Gate系统将设计的Y染色体基因进行合成，利用PhiC31重组酶整合系统将合成型基因整合到果蝇基因组上。通过对合成型基因的表达水平及合成型雄性果蝇育性进行检测，以确保合成型基因能够替代野生型基因发挥功能。同时，为了构建合成型Y染色体，我们开发了标签交替连续整合系统（SwMP-In）。如果Y染色体合成成功，这将是在多细胞生物中合成染色体的首例成功案例，首次打破了从低等生物到高等生物合成染色体的界限，为Y染色体的功能研究创造了前景。
The Y chromosome of Drosophila melanogaster is pivotal for male fertility and it contains ~40 Mb of DNA accounting for ~13% of the male genome. Despite the considerable size of the Y chromosome, the density of protein-coding sequences is extremely low. Only 16 genes reside on this chromosome, out of ~16,000 genes in the fly genome. The Y chromosome is nearly all heterochromatic and more than 70% of the Y chromosome is highly repetitive and consists of satellite DNA. Due to these features, and that most of the Y chromosome sequence is still missing from the genome sequence, the functions of the majority of genes on the Y chromosome remain elusive. More importantly, whether all the Y chromosome genes are necessary for the male fertility is not clear. To address this question, we employed the CRISPR/Cas9 system to generate mutations, and RNAi to interrogate most Y chromosome-encoded genes. In addition to the previously demonstrated role for kl-3 for male fertility, we demonstrate that two putative fertility genes kl-2 and kl-5 are also important for male fertility. In addition, we discovered that another gene, CCY also functions in male fertility. We demonstrate that mutation or RNAi knockdown of any of these four genes (CCY, kl-2, kl-3 and kl-5) disrupts nuclear elongation, and leads to defects in sperm individualization, including impairments in the individualization complex (IC) and synchronization. Our work establishes functions of additional Y-chromosome genes for male fertility, and indicates that not all Y chromosome genes are required for male fertility. Moreover, to learn more functions about Y chromosome, such as whether there exist some functional elements in the non-coding regions of the Y chromosome; whether the fertility genes are sufficient for male fertility, we employed synthetic biology to synthesize a syn-Y chromosome for researching. We designed and synthesized the Y genes based on the Golden Gate method, and generated synthetic flies depends on the PhiC31 site-specific recombination system. In order to seamless build a synthetic Y chromosome array, we developed a SwMP-In system. If successful, this will be the first time to synthesize chromosome in multicellular organism.
