鸡蛋壳超微结构与晶体结构的测定及全基因组关联分析

发布时间：2018-02-12 20:04

本文关键词： 蛋壳超微结构晶体结构 X射线晶体衍射 GWAS　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：蛋壳为胚胎生长发育提供了一个相对独立和稳定的内部环境,同时也是胚胎发育所需钙离子的来源。品质好的蛋壳在一定程度上能够降低食品安全的风险,也能减少由于蛋壳破损导致的经济损失,提高经济效益。同时蛋壳的形成过程也是一种经典的生物矿化模型。本研究使用鸡的600K高密度SNP芯片,对白来航与东乡绿壳蛋鸡正反交得到的F2群体进行SNP分型,使用扫描电镜与X射线晶体衍射两种方法,测定蛋壳超微结构与晶体结构,阐释其遗传基础,并探讨蛋壳超微结构与晶体结构之间的关系。选择F2群体927只母鸡进行基因分型,每只鸡收集66周龄的1个鸡蛋,取鸡蛋赤道部位蛋壳,使用扫描电镜测定蛋壳厚度(EST)、有效层厚度(ET)、乳突层厚度(MT)与乳突密度(MD)。使用混合线性模型进行全基因组关联分析。结果显示,EST、ET、MT与MD的表型变异系数分别为12.17%、14.83%、20.44%与23.83%,基于全基因组SNP估计的遗传力分别为0.39、0.36、0.17 与 0.19。719、784、1、10 个 SNP 分别与 EST、ET、MT、MD 显著相关。与 EST 和 ET 显著关联的SNP位于1号染色体的59.4Mb至68.5Mb区间,与MD显著关联的SNP位于6号染色体的20.6Mb至21.4Mb区间,与MT显著关联的SNP位于9号染色体的4.9Mb位置。ABCC9,KCNJ8,ITPR2和WNK1四个候选基因与ET和EST显著相关,它们均通过子宫组织的离子转运系统参与到蛋壳的形成过程。MT一方面受到乳突与乳突之间空间的影响;另一方面受到ITM2C的调控,其可能的作用方式是通过ITM2C-RIT2-Calmodulin-ITPR蛋白质之间的相互作用链条实现其对MT的调控。MD受到KNDC1的调控,其可能的作用方式有两种:其一是直接的方式,KNDC1与纤维核蛋白直接相互作用;其二是间接方式,KNDC1参与信号转导调控其它修饰蛋白来修饰纤维核蛋白。使用Rigaku R-AXIS SPIDERX射线晶体衍射仪测定晶体参数,获得前9个主要衍射峰的积分强度、总积分强度(TA)与晶体取向度(OD)。使用混合线性模型进行全基因组关联分析。结果显示,TA与蛋壳超微结构性状及蛋壳常规品质性状存在正相关关系,与OD之间存在负相关,说明颗粒越大的晶体,取向度越低,越倾向于随机分布。TA与OD的表型变异系数分别为9.4%与23.72%,基于全基因组SNP估计的遗传力分别为0.23与0.06。位于1号染色体55.7～69.3 Mb区间的621个SNP与TA显著相关。根据它们对应基因的功能及其mRNA与蛋白质在子宫组织细胞中的表达量,本研究认为DERA基因对蛋壳晶体的生长调节起到了重要作用。其作用途径可能有3种:一是为子宫组织细胞基质蛋白的合成及其与离子的转运过程中提供能量;二是降低高Ca2+浓度应激压力,保持细胞活性,并为蛋壳晶体的生长稳定地提供所需的Ca2+;三是通过调控子宫液中ATP的浓度,对蛋壳晶体的生长产生作用。
[Abstract]:Eggshells provide a relatively independent and stable internal environment for embryonic growth and development, and are also a source of calcium for embryonic development. Good quality eggshells can reduce the risk of food safety to some extent. It can also reduce the economic loss caused by eggshell breakage and improve economic benefit. The formation process of eggshell is also a classical biomineralization model. This study used 600K high density SNP chip of chicken. SNP typing was carried out on F2 population of white Laihang and Dongxiang green shell laying hens. The ultrastructure and crystal structure of egg shell were determined by scanning electron microscope and X-ray crystal diffraction, and the genetic basis was explained. The relationship between the ultrastructure of egg shell and crystal structure was studied. 927 hens from F2 population were selected for genotyping. One egg aged 66 weeks was collected from each hen and the egg shell was collected at equatorial part of the egg. The thickness of egg shell was measured by scanning electron microscope (SEM), the thickness of effective layer (et), the thickness of mastoid (MTT) and mastoid density (MDN) were measured by scanning electron microscope (SEM). The whole genome association analysis was carried out using mixed linear model. The results showed that the phenotypic variation coefficients between ESTT and MD were 12.17 ~ 14.83% and 23.83%, respectively. The heritability estimated based on genomic SNP was 0.39 / 0.36 / 0.17 and 0.19.719 / 778 / 4 / 10 SNP, respectively, which were significantly correlated with the MTMD of EST and et. The SNP with significant association with EST and et was located between 59.4 Mb and 68.5 Mb on chromosome 1. The SNP significantly associated with MD was located between 20.6Mb and 21.4Mb on chromosome 6, and the SNP significantly associated with MT was located at 4.9Mb on chromosome 9. The four candidate genes, ABCC9KCNJ8ITPR2 and WNK1, were significantly correlated with et and EST. They are involved in the formation of eggshell through the ion transport system of uterine tissue. MT is affected by the space between mastoid and mastoid on the one hand, and regulated by ITM2C on the other. Its possible mode of action is to realize the regulation of MT through the interaction chain between ITM2C-RIT2-Calmodulin-ITPR proteins. MD is regulated by KNDC1. There are two possible modes of action: one is the direct interaction between KNDC1 and fibronectin, one is the direct interaction between KNDC1 and fibronectin; The other is the indirect way that KNDC1 participates in signal transduction regulation of other modified proteins to modify fibronectin. The crystal parameters are measured by Rigaku R-AXIS SPIDERX crystal diffractometer, and the integral intensity of the first nine main diffraction peaks is obtained. Total integral strength (TAA) and crystal orientation were analyzed by using mixed linear model. The results showed that there was a positive correlation between TA and eggshell ultrastructure and egg shell quality, but a negative correlation was found between TA and OD. The larger the particle size, the lower the orientation. The coefficient of variation of phenotypic variation for random distribution of .TA and OD were 9.4% and 23.72, respectively, and the heritability estimated based on genomic SNP were 0.23 and 0.06.621 SNP located in the interval of 55.7mb and 69.3Mb on chromosome 1 were significantly correlated with TA. The function of the gene and the expression of mRNA and protein in uterine tissue cells, In this study, we believe that DERA gene plays an important role in the growth regulation of eggshell crystals. There may be three ways of its action: one is to provide energy for the synthesis of matrix protein and its transport with ions in uterine tissue cells; The second is to reduce the stress pressure of high concentration of Ca2, to maintain cell activity, and to provide the necessary Ca2 for the growth of eggshell crystal stably, and the third is to regulate the concentration of ATP in uterine fluid to produce effect on the growth of eggshell crystal.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S831

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