【作者】 李明辉；

【导师】 王德寿；

【作者基本信息】 西南大学 ， 动物学， 2014， 博士

【摘要】 行有性生殖的生物都有两种不同的性别,发育过程中都涉及到性别决定。因此,性别决定与分化及其分子机制是最基础的生物学问题之一。哺乳类性别决定与分化是由一系列转录因子和信号传导因子连接成网构成的协同作用通路,而硬骨鱼类还不清楚。许多经济动物(养殖鱼类)生长速率有明显的性别差异,如尼罗罗非鱼(Oreochromis niloticus)雄鱼比雌鱼生长快50%,单性鱼养殖具有较高的经济价值,因此养殖鱼类性别决定与分化的分子机制及性别控制成为水产动物学研究的热点,可以兼顾基础和应用研究。随着大片段基因组文库的构建和高通量DNA测序技术的发展,罗非鱼基因组序列已经公布,但缺乏Y染色体信息,有必要构建含Y染色体信息的大片段基因组文库。长期以来,养殖鱼类缺乏有效的基因敲除手段严重阻碍了其性别决定和分化分子机制的研究。近几年,人工核酸内切酶介导的基因组编辑技术TALEN和CRISPR/Cas9的出现使我们能对任意物种的基因组进行定点编辑。因此,本研究将探索在罗非鱼建立这两种基因敲除技术,并用于对dmrt1、foxl2、igf3和nanos等基因敲除,研究它们在性别决定与分化中的作用及分子机制。主要研究结果如下：1.以pCC2FOS为载体构建罗非鱼XY个体Fosmid基因组文库,共挑选115200个单菌落,保存在300块384孔板中,构建4800个行池、7200个列池、300个板池和25个超级池,形成微阵列,并对其进行复制备份。该文库插入片段平均长度为40kb左右,覆盖约罗非鱼基因组的3倍。连续传代实验表明,文库具有良好的稳定性。通过构建超级池—板池—行、列池三级池系统形成微阵列,能快速、准确、有效的筛选目的基因,仅需77个PCR反应(超级池25+板池12+行池16+列池24)就能筛到含有目的基因的克隆,解决了普遍存在的文库筛选难题。通过该文库尝试筛选了20个与性别分化相关基因,均获得2-5个阳性克隆,说明文库具有较好的实用性。罗非鱼XY基因组大片段文库的构建,为性别特异分子标记的筛选、性别决定候选基因的克隆、基因组编辑和遗传育种等研究提供了基础。2. TALEN基因组编辑技术已成功运用于多种模式生物。本研究在非模式生物罗非鱼中建立TALEN靶向基因敲除技术,它能高效的对dmrt1和foxl2进行敲除,且最高敲除率达到81%,亚克隆测序证实靶位点产生多种删除或插入不同数目碱基的突变类型。此外,TALEN诱导的高突变率个体在当代(G0)就能产生明显的表型。通过常规组织学、免疫组化、real-time PCR和激素测定等方法检测了Dmrt1和Foxl2缺失对性腺性别分化及相关基因表达的影响,结果显示：XY个体Dmrt1缺失使精巢不能正常发育,如输精管发育异常,精原细胞退化,甚至没有生殖细胞,类固醇生成细胞增生等。Dmrt1缺失的XY个体无性逆转发生。此外,与对照组精巢相比,Dmrt1缺失使精巢中foxl2、雌激素合成酶Cyp19a1a/cyp19a1a和血清雌激素(17β-雌二醇)水平升高。相反,XX个体Fox12缺失使卵母细胞退化,Cyp19a1a/cyp19a1a和血清雌激素水平显著降低；更重要的是,部分Fox12缺失的XX个体发生性逆转,并伴随Dmrt1和雄激素合成酶Cyp11b2的表达。该研究证实TALEN能高效应用于养殖鱼类罗非鱼的基因敲除；Dmrt1和Foxl2在性腺中拮抗性的参与罗非鱼性别分化,它们主要通过调控Cyp19ala的表达和雌激素的水平来影响性别分化。3.新兴的CRISPR/Cas9已在多个模式生物中证实能有效进行基因组编辑。本研究采用CRISPR/Cas9成功在非模式生物罗非鱼进行nanos2、nanos3、dmrt1和foxl2的敲除,最高突变率达到95%。CRISPR/Cas9诱导的基因突变在当代(G0)也能产生明显的表型。通过GFP-vasa’3UTR标记和生殖细胞特异表达Vasa染色证实,XY性腺缺失Nanos2和XX个体性腺腺缺失Nanos3均导致生殖细胞缺失,且性腺成中空管状结构。免疫组化结果显示Nanos2缺失的XY精巢仍然表达Dmrt1和Cyp11b2;而Nanos3缺失的XX性腺体细胞雄性化,表达Dmrt1和Cyp11b2,司时伴随着敲除鱼血清雄激素(11-KT)的上调和雌激素的下调。该结果表明无论基因型如何,生殖细胞缺失均导致性腺体细胞的雄性化。此外,CRISPR/Cas9敲除Dmrtl和Foxl2所产生的表型与TALEN敲除结果一致。CRISPR/Cas9诱导的foxl2和dmrt1突变能通过生殖细胞高效传递到F1代。F1代中检测到移码和非移码的foxl2突变体,这与其XX亲本的突变类型相一致；然而,与dmrt1XY亲本突变类型不一致,敲除鱼的精子以及受精获得的F1代中仅检测到非移码的突变,这说明Dmrt1对罗非鱼精子发育和成熟具有重要作用。这些结果表明CRISPR/Cas9可用于罗非鱼基因的高效、快速编辑,而且是可遗传的。4.最近,本实验室发现一个仅在硬骨鱼类存在的igf3,它只在性腺表达,然而,它在性腺发育过程中的表达模式、转录调控及在性腺分化中的作用还有待阐明。我们通过real-timePCR检测发现igf3在孵化后5天到孵化后40天卵巢的表达量高于精巢,而孵化后50天到孵化后70天低于精巢的表达。通过制备Igf3多克隆抗体,免疫组化检测显示Igf3在孵化后10天到成体雌雄性腺的体细胞中表达。利用Igf3重组蛋白处理原代培养的卵巢和精巢细胞,real-time PCR检测发现Igf3以时问、剂量依赖的方式增强性腺分化相关转录因子foxl2、 dmrt1、 nr5a1(sfl)和类固醇酶cyp19a1a、cyp11b2、cyp11a1、hsd3b2和cyp17a1的表达。通过CRISPR/Cas9成功在XX、XY罗非鱼敲除igf3基因,单条鱼的突变率最高达到95%。Igf3缺失导致3月龄XY性腺中无精母细胞和精细胞、但存在精原细胞,精巢只有少量Cyp11b2表达；而对照组XY精巢存在各级生精细胞,且有大量Cyp11b2表达。Real-time PCR结果显示：敲除鱼性腺dmrt1、nr5a1和cyp11b2mRNA的表达水平也明显低于对照鱼。与此相吻合,激素测定进一步证实敲除鱼血清雄激素水平明显低于对照鱼。另一方面,Igf3缺失导致3月龄XX卵巢中存在大量卵原细胞,极少的Ⅰ、Ⅱ时相卵母细胞,卵巢只有少量Cypl9ala表达；而对照组XX卵巢已有大量各时相卵母细胞,只有边缘存在少量卵原细胞,卵巢中大量的Cyp19a1a表达于间质细胞。Real-time PCR结果也显示：敲除鱼foxl2、nr5a1和cyp19a1a mRNA的表达水平也明显低于对照鱼。与此相吻合,激素测定进一步证实敲除鱼血清雌激素水平明显低于对照鱼。这些体外和体内研究都表明Igf3参与调控性别分化相关转录因子和类固醇酶的表达,且Igf3参与调控罗非鱼生殖细胞的减数分裂。最后,利用Fosmid文库获得的igf3启动子序列,构建双荧光素酶报告基因载体在HEK293细胞中进行启动子分析发现：igf3能直接被Nr5a1(Sfl)激活,而Fox12、NrOb1a (Dax1)和NrOblb (Dax2)单独转染没有作用,但与Nr5al共转染时均能增强Nr5al激活的igf3转录活性；然而,Dmrt1、雌激素在与受体共转染时抑制igf3的转录。Forskolin能增强Nr5al和igf3的转录。此外,igf3在Dmrt1和Foxl2TALEN敲除鱼的表达升高。这些结果表明Igf3能调控foxl2、dmrtl、nr5a1和类固醇合成酶的表达,反之,igf3的表达又受到Foxl2、Dmrt1、Nr5a1和雌激素的调控,它们之间形成紧密的调控环作用于罗非鱼生殖细胞的减数分裂。综上所述：本研究建立了罗非鱼微阵列Fosmid基因组文库、TALEN和CRISPR/Cas9靶向基因敲除技术,通过TALEN和CRISPR/Cas9研究了Dmrt1、Foxl2、Nanos2、Nanos3以及新发现的Igf3在硬骨鱼类性别决定与分化的功能,证明其在研究硬骨鱼类性别决定与分化的分子机制方面具有独特的优势。目前这些技术已广泛用于本实验室罗非鱼基因筛选和基因编辑。TALEN和CRISPR/Cas9技术的建立为养殖鱼类的基因功能以及性别控制研究提供有效的技术手段。更多还原

【Abstract】 Clafrication the sex determination and differentiation of organisms with sexual reproducation is one of the most basic biological issues. Growth rates of many economic animals, such as Nile tilapia Oreochromis niloticus, showed significant sexual dimorphism, with the male grow faster than the female by50%. The molecular mechanism of tilapia sex determination and sex control has become the hot issue of aquaculture because of results from tilapia are useful for both basic research and aquaculture. With the development of genomic library and high-throughput DNA sequencing technology, the genome sequence of tilapia are available. The main challenge is to interpret the function of these sequences. For a long time, the lack of effective methodology of gene knockout seriously hampered the studies of tilapia sex determination. Recently, genome editing nucleases (TALEN) and the CRISPR/Cas9system have provided two powerful approaches to explore any gene related to sex determination. In the present study, TALEN and CRISPR/Cas9were established in tilapia and subsequently dmrtl, foxl2, the newly discovered igf3and nanos were mutated these technologies so as to elucidate the function of these genes in tilapia sex determination and differentiation. The main results are listed as follows:1) A high-quality Fosmid library of the XY Nile tilapia was successfully constructed, microarrayed and characterized using the vector pCC2FOS. It encompasses115200non-redundant clones with an average insert size of40kb and3-fold genome coverage. All clones were arrayed in300plates of384-well plate. For each plate, column-, row-and plate-pools were constructed, and therefore, totally4800column pools,7200row pools and300plate pools were constructed and arrayed. Finally,25super pools were made with each covering twelve384-well plates. Stability of the library was tested by100generation passage experiment. No loss or rearrangement of the inserted DNA fragment was found during the continuous passage. Any targeted gene can be fast screened only by minimally77colony PCR due to the array of super-pools, plate-pools, row-and column-pools. Twenty genes related to sex differentiation and growth were successfully isolated from this library, with each having2-5positive clones. The Fosmid library will be helpful to isolate sex linked marker, sex determining gene, genome editing and genetic and breeding.2) Transcription activator-like effector nucleases (TALENs) have been proved to be effective in several organisms genome editing. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Two pairs of TALENs were constructed to target genes related to sex differentiation, including dmrtl,foxl2, and both resulted in indel mutations with maximum efficiencies of up to81%at the targeted loci. Effects of dmrtl and foxl2mutation on gonadal phenotype, sex differentiation and related gene expression were analyzed by histology, immunohistochemistry and real-time PCR. In Dmrtl deficiency testes, phenotypes of significant testicular regression, including deformed efferent duct, degenerated spermatogonia or even a complete loss of germ cells, and proliferated steroidogenic cells, were observed. In addition, disruption of Dmrtl in XY fish resulted in increased foxl2, cyp19ala expression, serum estradiol-17β and11-ketotestosterone levels. On the contrary, deficiency of Foxl2in XX fish exhibited varying degrees of oocyte degeneration, significantly decreased aromatase gene expression and serum estradiol-17β levels. Some Foxl2deficiency fish even exhibited complete sex reversal with high expression level of Dmrtl and Cyp11b2. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2and Dmrtl play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.3) CRISPR/Cas9system has recently become powerful tool for targeted genome editing in model animals. Here, we report that disruption of selected genes, including nanos2, nanos3, dmrtl and foxl2, in tilapia through CRISPR/Cas9system was achieved with high efficiency (up to95%). Furthermore, obvious phenotypes were observed in GO generation after mutation of germ cell or somatic cell specific genes. For example, loss of Nanos2and Nanos3in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, and masculinization of somatic cells in both XY and XX gonads as demonstrated by Dmrtl and Cyp11b2immunohistochemistry and by up-regulation of serum androgen levels. In addition, mutations in dmrtl and foxl2induced by CRISPR/Cas9were efficiently transmitted through the germline to F1. Mutations were found to be transmitted to their F1progeny with a rate of22.2%(4of18, dmrtl) and58.3%(10of24, foxl2), respectively. The F1foxl2larvae carried deletion mutations including in-frame and frame-shift deletions as their GO founders. In contrast, the F1dmrtl larvae only carried3or21bp in-frame deletions, the same as found in the sperms used for fertilization but different from the GO founders which carried both in-frame and frame-shift deletions. Therefore, frame-shift deletions in Dmrtl in tilapia germ cells probably affect their development, meiosis, and maturation in tilapia. Taken together, our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing an effective approach for generating loss-of-function mutants.4) Previous studies from our lab have reported that a novel igf3, which is distinct from the conventional igfl and igf2, is only expressed in fish gonads, thus implying its special roles in gonad sex differentiation. In this study, igf3mRNA expression in the tilapia ovary was found to be higher than in the testis from5to40days after hatching (dah), but was lower than the testis from50to70dah. Consistently, Igf3protein signal was detected in the somatic cells of the XX and XY gonads from10dah till adulthood by immunohistochemistry using a specific Igf3polyclonal antibody. Incubation of ovarian and testicular cells in primary culture with recombinant Igf3significantly increased nr5a1,foxl2, dmrtl, cyp19a1a, cyp11a1, cyp11b2, and hsd3b2and cyp17a1expression in time-and dose-dependent manners. Igf3was successfully mutated by CRISPR/Cas9in XX and XY tilapia with high efficiency reach at95%. Histology examination showed Igf3deficiency testis have almost no spermatocyte and sperm, but with spermatogonia, and weak Cyp11b2expression detected. Spermatogonia, spermatocyte and sperm existed in the3month control XY testis, which expressed strong Cyp11b2. Real-time PCR results showed that expression level of dmrtl, nr5al and cypllb2were decreased in Igf3deficiency testis, compared with the control. Consistent with these results, knockout of Igf3in the XY fish resulted in decreased level of serum11-KT compared with the control fish. On the other hand, a large number of oogonia, little phage I and II oocyte was observed in the3months Igf3deficiency ovary, which expressed low Cypl9ala. In control XX ovary, little oogonia, different stage of I, II, III oocyte was observed, and Cypl9ala was highly expressed in the interstital cells of3months control ovary. Real-time PCR results showed that expression level of foxl2, nr5a1and cypl9ala were down regulated after loss of Igf3, compared with the control. Consistent with these results, knockout of Igf3in the XX fish resulted in decreased level of E2compared with the control fish. These results implied the gonad specific Igf3take part in teleost meiosis through regulating transcription factors and steroids. On the other hand, the igf3promoter sequence was obtained from Fosmid library. Promoter analysis using luciferase assays in HEK293cells revealed that igf3promoter activity was directly activated by Nr5a1(Sf1), and further enhanced by Foxl2, NrObla (Dax1) and NrOblb (Dax2) but repressed by Dmrtl and estrogen receptor along with17P-estradiol treatment. In addition, igf3promoter activity was increased slightly by Forskolin treatment alone, but synergistically up-regulated by transfection with nr5a1. Consistent with the results from promoter analysis, igf3expression was up-regulated after Dmrtl and Foxl2deficiency. These in vitro results correlated well with the expression profile of igf3during early gonad differentiation. Our results indicated that igf3is involved in fish gonad steroidogenesis because of its ability to regulate the expression of foxl2, dmrtl, nr5a1and steroidogenic enzymes. The expression of igf3is in turn regulated by the transcription factors Foxl2, Dmrtl and Nr5al as well as by17β-estradiol treatment.In summary, microarrayed Fosmid library, TALEN and CRISPR/Cas9were successfully established in a worldwide cultured fish, Nile tilapia. The function of Dmrtl, Foxl2, Nanos2, Nanos3and Igf3in teleost sex determination and differentiation was studied by TALEN and CRISPR/Cas9, which showing the advantages of these two technologies to elucidate the molecular mechanisms of teleost sex determination and differentiation. The establishment of these platforms promotes the studies of functional genomics research and sex control in aquaculture.更多还原