本文關(guān)鍵詞： 蛋殼 超微結(jié)構(gòu) 晶體結(jié)構(gòu) X射線晶體衍射 GWAS　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：蛋殼為胚胎生長發(fā)育提供了一個相對獨(dú)立和穩(wěn)定的內(nèi)部環(huán)境,同時也是胚胎發(fā)育所需鈣離子的來源。品質(zhì)好的蛋殼在一定程度上能夠降低食品安全的風(fēng)險,也能減少由于蛋殼破損導(dǎo)致的經(jīng)濟(jì)損失,提高經(jīng)濟(jì)效益。同時蛋殼的形成過程也是一種經(jīng)典的生物礦化模型。本研究使用雞的600K高密度SNP芯片,對白來航與東鄉(xiāng)綠殼蛋雞正反交得到的F2群體進(jìn)行SNP分型,使用掃描電鏡與X射線晶體衍射兩種方法,測定蛋殼超微結(jié)構(gòu)與晶體結(jié)構(gòu),闡釋其遺傳基礎(chǔ),并探討蛋殼超微結(jié)構(gòu)與晶體結(jié)構(gòu)之間的關(guān)系。選擇F2群體927只母雞進(jìn)行基因分型,每只雞收集66周齡的1個雞蛋,取雞蛋赤道部位蛋殼,使用掃描電鏡測定蛋殼厚度(EST)、有效層厚度(ET)、乳突層厚度(MT)與乳突密度(MD)。使用混合線性模型進(jìn)行全基因組關(guān)聯(lián)分析。結(jié)果顯示,EST、ET、MT與MD的表型變異系數(shù)分別為12.17%、14.83%、20.44%與23.83%,基于全基因組SNP估計的遺傳力分別為0.39、0.36、0.17 與 0.19。719、784、1、10 個 SNP 分別與 EST、ET、MT、MD 顯著相關(guān)。與 EST 和 ET 顯著關(guān)聯(lián)的SNP位于1號染色體的59.4Mb至68.5Mb區(qū)間,與MD顯著關(guān)聯(lián)的SNP位于6號染色體的20.6Mb至21.4Mb區(qū)間,與MT顯著關(guān)聯(lián)的SNP位于9號染色體的4.9Mb位置。ABCC9,KCNJ8,ITPR2和WNK1四個候選基因與ET和EST顯著相關(guān),它們均通過子宮組織的離子轉(zhuǎn)運(yùn)系統(tǒng)參與到蛋殼的形成過程。MT一方面受到乳突與乳突之間空間的影響;另一方面受到ITM2C的調(diào)控,其可能的作用方式是通過ITM2C-RIT2-Calmodulin-ITPR蛋白質(zhì)之間的相互作用鏈條實(shí)現(xiàn)其對MT的調(diào)控。MD受到KNDC1的調(diào)控,其可能的作用方式有兩種:其一是直接的方式,KNDC1與纖維核蛋白直接相互作用;其二是間接方式,KNDC1參與信號轉(zhuǎn)導(dǎo)調(diào)控其它修飾蛋白來修飾纖維核蛋白。使用Rigaku R-AXIS SPIDERX射線晶體衍射儀測定晶體參數(shù),獲得前9個主要衍射峰的積分強(qiáng)度、總積分強(qiáng)度(TA)與晶體取向度(OD)。使用混合線性模型進(jìn)行全基因組關(guān)聯(lián)分析。結(jié)果顯示,TA與蛋殼超微結(jié)構(gòu)性狀及蛋殼常規(guī)品質(zhì)性狀存在正相關(guān)關(guān)系,與OD之間存在負(fù)相關(guān),說明顆粒越大的晶體,取向度越低,越傾向于隨機(jī)分布。TA與OD的表型變異系數(shù)分別為9.4%與23.72%,基于全基因組SNP估計的遺傳力分別為0.23與0.06。位于1號染色體55.7～69.3 Mb區(qū)間的621個SNP與TA顯著相關(guān)。根據(jù)它們對應(yīng)基因的功能及其mRNA與蛋白質(zhì)在子宮組織細(xì)胞中的表達(dá)量,本研究認(rèn)為DERA基因?qū)Φ皻ぞw的生長調(diào)節(jié)起到了重要作用。其作用途徑可能有3種:一是為子宮組織細(xì)胞基質(zhì)蛋白的合成及其與離子的轉(zhuǎn)運(yùn)過程中提供能量;二是降低高Ca2+濃度應(yīng)激壓力,保持細(xì)胞活性,并為蛋殼晶體的生長穩(wěn)定地提供所需的Ca2+;三是通過調(diào)控子宮液中ATP的濃度,對蛋殼晶體的生長產(chǎn)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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S831

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