【作者】 杨爱玲；

【导师】 张燕君；

【作者基本信息】 山东大学 ， 发育生物学， 2017， 硕士

【摘要】 生命的延续和发展依赖于生物的繁殖。有关生殖发育的研究受到越来越多的关注。斑马鱼作为一种常用的模式生物,受精和发育均在体外进行,方便研究。除此之外,作为脊椎动物,斑马鱼的基因同人类基因的功能相似,组织器官的结构同源。因此,在现代生命科学研究的各个方面广泛使用。腺苷酸激酶(Adenylate kinase,AK)是遍及于植物、动物和微生物体内的一种单体酶,在需要高水平腺苷三磷酸(Adenosine triphosphate,ATP)的细胞中大量存在。近年来,人们在细胞凋亡过程中发现有AK的迁移和酶活的变化,且AK缺陷与人类很多疾病的发生都有直接关系。有文献报道,人的AK1-4、6-7均表达于睾丸和附睾,AK5仅表达于附睾;AK6还表达于精子尾部。但在雄性生殖系统中AK的作用目前知道的还很少。实验室合作者前期研究发现,在人类弱精症患者中,AK相关基因表达异常。因此,本论文采用CRISPR/Cas9技术,敲除斑马鱼ak4,研究其对雄性生殖系统中ATP含量和AMP/ATP比例的影响以及对雄性生殖细胞凋亡的影响,为进一步治疗与此相关的疾病提供理论支持。通过序列比对和进化分析,发现斑马鱼各Ak彼此之间的序列一致性仅为8.5%,说明斑马鱼ak家族基因在序列、功能上有很大分化。qPCR技术分析斑马鱼各ak在胚胎发育早期和成体各组织器官的表达,发现斑马鱼ak7a既有有母源表达,又有合子表达;其他ak以合子表达为主;在精巢中表达的ak有ak1、ak4、ak6、ak7a、ak7b、ak8和ak9。由此明确了 ak各亚型在斑马鱼的表达情况,为针对特定的功能研究进行基因敲除提供了线索。随后采用CRISPR/Cas9技术,成功敲除斑马鱼腺苷酸激酶ak4基因,并筛选到纯合突变体后代。用HPLC方法测定精巢中ATP、ADP、AMP的含量及AMP/ATP比例,结果表明,突变体精巢中ATP含量显著减少,AMP含量未发生明显变化,AMP/ATP比值升高。说明精巢中细胞的能量状态发生了显著变化。用TUNEL和FCM检测,发现突变体精巢中细胞凋亡增多。结合其他文献和本实验结果,推测细胞中这种能量变化导致了 ak4突变体雄性生殖细胞凋亡。在8 h、24 h分别检测,都发现ak4雄性突变体后代存活率下降,死亡率上升,推测是由于ak4突变后,细胞凋亡增加导致精子质量下降所致。突变体胚胎在发育早期下包慢,卵黄大,色素浅,身体短,发育滞后。结合ak4在早期发育中的表达,这显示ak4也参与了早期胚胎发育过程。另外,还采用CRISPR/Cas9敲除了斑马鱼ak6和ak7a基因,制备了 F0代突变体。更多还原

【Abstract】 The continuation and development of life rely on the breeding of creatures.Increasing attentions have been paid toward research of reproductive development.As a commonly used animal model,their fertilization and development are carried out in vitro;this makes them be easy to handle for the experiments.In addition,as a vertebrate,the genome of zebrafish is functionally similar to the human,and the structure of the tissues and organs is homologous.Therefore,zebrafish is broadly used in various aspects of life science research.Adenylate kinase(AK)is a monomeric enzyme being prevalent in microbes,plants and animals and is abundant in cells that require high levels of Adenosine triphosphate(ATP).Recently,the migration and alteration of enzyme activity of AK have been observed in the course of cellular apoptosis,and the metabolic signal defects of AK are directly related to many human diseases.It was reported that human AK1-4,6-7 are expressed in the testis and epididymis,AK5is only expressed in the epididymis,and AK6 is also expressed in the sperm tail.But the role of AK in the male reproductive system still remains unclear.Previous study from our collaborators found that AK genes abnormally express in patients with weak schistosomiasis.Hereto,in the study CRISPR/Cas9 technique was used to knock out zebrafish ak4 gene to invest its effect on AMP/ATP ratio in male reproductive system and its effect on male germ cell development and reproductive capacity,to provide theoretical support for further treating relevant diseases.Through sequence alignment and evolutionary analysis,it was found that the sequence identity of each Ak gene in zebrafish was merely 8.5%,indicating that the ak gene family of zebrafish had a great differentiation in sequence and function.The expression of each ak gene in various tissues or organs in the early stage of embryotic development and adult was analyzed by using qPCR assay,indicating that zebrafish ak7a has not only maternal expression,but also zygotic expression;The other ak genes are mainly zygotic expression;akl,ak4,ak6,ak7a,ak7b,ak8 and ak9 are expressed in the testis.This clarifies the expression status of ak subtypes in zebrafish and provides clues to perform gene knockout for specific functional studies.Then by using CRISPR/Cas9 technology,the zebrafish adenylate kinase ak4 gene was successfully knocked out,followed by screening to homozygous mutant offspring.The content of ATP,ADP,AMP and AMP/ATP ratio in the testis were determined by HPLC.The results showed that the content of ATP in the mutant was significantly decreased,the content of AMP did not change significantly,and the AMP/ATP ratio increased.This Indicated that the energy state of the cells in the testes significantly changed.The apoptosis was increased in testis of mutant zebrafish detected by TUNEL and flow cytometry assay(FCM).Based on the results of other literatures and this study,it is speculated that the energy change in cells led to the apoptosis of ak4 mutant male germ cells.At 8 h and 24 h,it was found that the survival rate of ak4 male mutant was decreased and the mortality rate was increased.It was presumably because that the increase of apoptosis caused the decrease of sperm quality after ak4 mutation.In the early development of mutant embryos,the yolk got large,the pigment was shallow,and the body was short,and the development got lagged.In the combination of the expression of ak4 in early development,suggesting that ak4 is also involved in early embryonic development.In addition,we also knock out ak6 and ak7a genes in zebrafish by using CRISPR/Cas9 technique,and generated FO mutant zebrafish.更多还原