本文選題：雛鴕鳥(niǎo) + 硼�。� 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：非洲鴕鳥(niǎo)具有很大的經(jīng)濟(jì)價(jià)值和科學(xué)價(jià)值。鴕鳥(niǎo)育雛期長(zhǎng)達(dá)3個(gè)月,人工養(yǎng)殖的鴕鳥(niǎo)容易受到應(yīng)激、感染和疾病的影響,使得育雛期鴕鳥(niǎo)的發(fā)病率和死亡率高達(dá)10%-50%。此外,鴕鳥(niǎo)是快速生長(zhǎng)的鳥(niǎo)類,育雛期鴕鳥(niǎo)個(gè)體發(fā)育差別較大,免疫機(jī)能的強(qiáng)弱直接反應(yīng)了育雛期鴕鳥(niǎo)生長(zhǎng)發(fā)育狀況。胸腺是機(jī)體內(nèi)重要的中樞免疫器官和淋巴器官,也是機(jī)體抵抗外界抗原刺激,參與機(jī)體T淋巴細(xì)胞分化的重要免疫器官。硼是動(dòng)物體必需的微量元素,具有多種生物學(xué)功能。硼在免疫系統(tǒng)中發(fā)揮重要的調(diào)節(jié)作用,適量的硼有利于免疫器官的生長(zhǎng)發(fā)育。本試驗(yàn)選取1日齡的雛鴕鳥(niǎo)作為研究對(duì)象,飲水中添加梯度劑量的硼酸,飼喂至90日齡,宰殺取胸腺。研究硼對(duì)鴕鳥(niǎo)中樞免疫器官胸腺的調(diào)節(jié)作用,闡釋硼參與鴕鳥(niǎo)機(jī)體免疫應(yīng)答的作用機(jī)制。1.硼對(duì)雛鴕鳥(niǎo)胸腺生長(zhǎng)發(fā)育相關(guān)基因的影響Foxn1參與胸腺上皮細(xì)胞的分化發(fā)育過(guò)程,并發(fā)揮關(guān)鍵調(diào)控作用,BMP2和BMP4參與調(diào)控胸腺T細(xì)胞的發(fā)育,Foxn1,BMP2和BMP4基因與胸腺內(nèi)環(huán)境穩(wěn)態(tài)密切相關(guān)。研究不同水平的硼對(duì)鴕鳥(niǎo)胸腺細(xì)胞凋亡的影響及對(duì)鴕鳥(niǎo)胸腺發(fā)育相關(guān)基因Foxn1,BMP2和BMP4的表達(dá)調(diào)控,為揭示硼對(duì)鴕鳥(niǎo)胸腺發(fā)育影響的相關(guān)機(jī)制提供參考。HE染色技術(shù)觀察硼對(duì)雛鴕鳥(niǎo)胸腺組織學(xué)結(jié)構(gòu)的影響,RACE PCR克隆鴕鳥(niǎo)Foxn1基因的序列,免疫組織化學(xué)技術(shù)和western blot技術(shù)檢測(cè)Foxn1蛋白在鴕鳥(niǎo)胸腺內(nèi)的定位表達(dá)。免疫熒光技術(shù)檢測(cè)BMP2和BMP4蛋白在鴕鳥(niǎo)胸腺內(nèi)的表達(dá)。熒光定量PCR技術(shù)檢測(cè)Foxn1基因,BMP2基因和BMP4基因的mRNA表達(dá)水平。TUNEL技術(shù)檢測(cè)鴕鳥(niǎo)胸腺內(nèi)細(xì)胞凋亡情況,免疫組織化學(xué)檢測(cè)活化體caspase-3在鴕鳥(niǎo)胸腺內(nèi)的定位表達(dá)。實(shí)驗(yàn)結(jié)果如下:(1)HE染色觀察硼對(duì)雛鴕鳥(niǎo)胸腺組織學(xué)結(jié)構(gòu)的影響,高劑量硼組(B320組和B640組)胸腺的形態(tài)結(jié)構(gòu)不完整,皮髓交界分界不清楚,胸腺皮質(zhì)內(nèi)出現(xiàn)“星空狀”外觀,胸腺細(xì)胞急劇減少和空竭,胸腺髓質(zhì)內(nèi)淋巴細(xì)胞增多,胞核固縮深染,B640組胸腺胸腺結(jié)構(gòu)破壞最嚴(yán)重,胸腺上皮細(xì)胞空泡化,髓質(zhì)部最明顯。低劑量硼組(B40和B80)胸腺形態(tài)結(jié)構(gòu)與對(duì)照組基本一致。(2)TUNEL技術(shù)檢測(cè)雛鴕鳥(niǎo)胸腺內(nèi)細(xì)胞凋亡,與對(duì)照組相比,B80組和B160組胸腺內(nèi)凋亡細(xì)胞數(shù)量較少,B320組和B640組胸腺內(nèi)凋亡細(xì)胞數(shù)量增多,凋亡細(xì)胞成群分布。(3)Caspase-3活化體檢測(cè)發(fā)現(xiàn),B320組和B640組胸腺內(nèi)活化體caspase-3陽(yáng)性信號(hào)顯著增強(qiáng),高劑量的硼誘導(dǎo)caspase-3的活化,誘發(fā)鴕鳥(niǎo)胸腺細(xì)胞凋亡。(4)racepcr克隆擴(kuò)增鴕鳥(niǎo)foxn1基因,隨機(jī)引物擴(kuò)增鴕鳥(niǎo)foxn1基因中間保守片段,長(zhǎng)為1477bp,5’race和3’race的長(zhǎng)度分別為384bp和1050bp,全長(zhǎng)為2736bp,編碼654個(gè)氨基酸。鴕鳥(niǎo)foxn1基因是高度保守的基因,與獵隼、虎皮鸚鵡、游隼、石鴿、野鴨和雞的foxn1基因同源性分別為92.1%,91.1%,90.8%,89.6%,88.1%和83.5%。(5)foxn1陽(yáng)性信號(hào)主要分布在鴕鳥(niǎo)胸腺髓質(zhì)部,胸腺皮質(zhì)部有少量分布,主要標(biāo)記胸腺上皮細(xì)胞。80mg/l的硼酸劑量組能夠顯著提高foxn1蛋白在鴕鳥(niǎo)胸腺中的表達(dá),而640mg/l的硼酸劑量能夠顯著降低foxn1蛋白在胸腺中的表達(dá)。(6)bmp2和bmp4的mrna表達(dá)水平呈硼劑量依賴性,表達(dá)趨勢(shì)先上升后下降,在80mg/l的硼處理組中,bmp2和bmp4在鴕鳥(niǎo)胸腺內(nèi)顯著高表達(dá),在640mg/l的硼處理組中,bmp2和bmp4的表達(dá)水平被顯著抑制。飲水中添加適量的硼促進(jìn)雛鴕鳥(niǎo)胸腺的發(fā)育,鴕鳥(niǎo)胸腺正常發(fā)育;過(guò)量的硼導(dǎo)致雛鴕鳥(niǎo)胸腺組織學(xué)結(jié)構(gòu)破壞,誘發(fā)雛鴕鳥(niǎo)胸腺內(nèi)細(xì)胞凋亡,活化體caspase-3表達(dá)增強(qiáng),抑制雛鴕鳥(niǎo)胸腺的正常發(fā)育。80mg/l硼促進(jìn)bmp2,bmp4,foxn1蛋白的表達(dá),bmp2,bmp4,foxn1蛋白表達(dá)適度增加會(huì)促進(jìn)胸腺上皮細(xì)胞的分化發(fā)育,從而促進(jìn)t細(xì)胞的分化發(fā)育,增強(qiáng)鴕鳥(niǎo)機(jī)體免疫抵抗力。高劑量的硼(640mg/l)顯著抑制bmp2,bmp4,foxn1蛋白在鴕鳥(niǎo)胸腺內(nèi)的表達(dá),導(dǎo)致鴕鳥(niǎo)胸腺結(jié)構(gòu)的破壞和退化,胸腺的皮髓交界處損壞嚴(yán)重,胸腺上皮細(xì)胞分化發(fā)育受阻,抑制t細(xì)胞的正常生長(zhǎng)發(fā)育,降低鴕鳥(niǎo)機(jī)體免疫機(jī)能。2.雛鴕鳥(niǎo)胸腺應(yīng)答硼刺激的rna-seq分析為了深入研究硼參與鴕鳥(niǎo)免疫應(yīng)答的作用機(jī)制,本研究采用rna-seq技術(shù),首次對(duì)微量元素硼在鴕鳥(niǎo)胸腺免疫應(yīng)答反應(yīng)中的作用進(jìn)行了高通量測(cè)序分析。本試驗(yàn)共選取3個(gè)樣本進(jìn)行rna-seq分析,分別是對(duì)照組鴕鳥(niǎo)胸腺,b80組鴕鳥(niǎo)胸腺,b640組鴕鳥(niǎo)胸腺。rna-seq分析結(jié)果顯示:(1)3個(gè)鴕鳥(niǎo)胸腺測(cè)序文庫(kù)(control組樣本文庫(kù),b80組樣本文庫(kù)和b640組樣本文庫(kù)),分別包含3220173000,3427886800和3199872000條rawdata。control組樣品,b80組樣品和b640組樣品的cleanreads數(shù)分別為3175394800,3379919000和3157669200,cleandata占reads總數(shù)的比例分別為98.61%,98.60%和98.68%。(2)control組樣品,b80組樣品和b640組樣品單一比對(duì)上鴕鳥(niǎo)參考基因組的reads數(shù)分別為11929989,12851275和11411617,占總reads的比例分別為75.14%,76.04%和72.28%。(3)基因覆蓋度統(tǒng)計(jì)分析顯示,三個(gè)文庫(kù)中測(cè)序覆蓋度達(dá)90-100%的基因數(shù)量占總數(shù)量的比例分別為72%,71%和69%。(4)成對(duì)差異基因統(tǒng)計(jì)結(jié)果顯示:control組和b640組,b80組和b640組,control組和b80組差異基因比較總計(jì)分別有2044個(gè)(上調(diào)基因228個(gè),下調(diào)基因1816個(gè)),1085個(gè)(上調(diào)基因222個(gè),下調(diào)基因863個(gè))和902個(gè)(上調(diào)基因309個(gè),下調(diào)基因593個(gè))差異基因。(5)差異基因趨勢(shì)分析結(jié)果顯示:本試驗(yàn)中所有的差異基因共分成了7種表達(dá)趨勢(shì),其中趨勢(shì)0,趨勢(shì)1和趨勢(shì)3具有差異顯著性。趨勢(shì)0中共包含1290個(gè)差異基因,在3個(gè)文庫(kù)中(control,b80和b640)的表達(dá)依次呈現(xiàn)持續(xù)下降的趨勢(shì);趨勢(shì)1中共包含1030個(gè)差異基因,在3個(gè)文庫(kù)中的表達(dá)依次呈現(xiàn)先下降后保持不變的趨勢(shì);趨勢(shì)3中共包含1487個(gè)差異基因,在3個(gè)文庫(kù)中的表達(dá)依次呈現(xiàn)先上升或不變后下降的趨勢(shì)。(6)差異基因kegg信號(hào)通路富集分析結(jié)果顯示:富集前10條信通路中細(xì)胞因子-受體互作通路,癌癥通路,鈣離子信號(hào)通路,肌動(dòng)蛋白細(xì)胞骨架調(diào)節(jié)通路和mapk信號(hào)通路顯著富集,這些信號(hào)通路主要參與機(jī)體的免疫應(yīng)答及炎癥應(yīng)答過(guò)程。(7)成對(duì)樣品差異基因kegg富集結(jié)果顯示,除上述信號(hào)通路,硼還參與toll樣受體信號(hào)通路,b細(xì)胞受體通路,t細(xì)胞受體通路和凋亡通路的調(diào)節(jié)。由此表明,硼對(duì)鴕鳥(niǎo)機(jī)體的炎癥及免疫機(jī)能的相關(guān)信號(hào)通路的影響,主要包括mapk信號(hào)通路,鈣離子通路,b細(xì)胞受體通路,t細(xì)胞受體通路,肌動(dòng)蛋白調(diào)節(jié)信號(hào)通路,toll樣受體信號(hào)通路,凋亡信號(hào)通路及癌癥信號(hào)通路。3.硼對(duì)鴕鳥(niǎo)機(jī)體免疫、炎癥及生長(zhǎng)發(fā)育相關(guān)通路的影響為了進(jìn)一步驗(yàn)證rna-seq的分析結(jié)果,并探討不同劑量的硼對(duì)鴕鳥(niǎo)胸腺內(nèi)免疫機(jī)能相關(guān)信號(hào)通路的影響作用,本項(xiàng)目對(duì)參與免疫機(jī)能調(diào)節(jié)的信號(hào)通路進(jìn)行了研究,主要包括mapk通路,鈣離子通路,b細(xì)胞受體通路,t細(xì)胞受體通路,toll樣受體通路,癌癥信號(hào)通路以及細(xì)胞凋亡通路。(1)硼對(duì)mapk通路活性的影響:共有27個(gè)差異基因富集到mapk信號(hào)通路中,control組和b80組中的大部分基因表達(dá)上升,而b640組中的基因多數(shù)表達(dá)下調(diào),27個(gè)差異基因中有24個(gè)基因表達(dá)下降,3個(gè)基因表達(dá)上升。硼能夠影響鴕鳥(niǎo)機(jī)體內(nèi)ras/erk通路,jnk通路和p38mapk通路,但不能參與erk5通路的調(diào)節(jié)。qrt-pcr及westernblot驗(yàn)證結(jié)果顯示,80mg/l硼促進(jìn)了鴕鳥(niǎo)胸腺內(nèi)p-erk,p-jnk和p-p38蛋白水平的表達(dá),隨著硼劑量的增加,p-erk,p-jnk和p-p38蛋白表達(dá)水平逐漸降低,在640 mg/L硼組蛋白表達(dá)水平最低。硼能夠調(diào)節(jié)ERK,JNK以及p38MAPK通路中激酶的表達(dá)水平,影響MAPK信號(hào)通路的活性。(2)硼對(duì)鈣離子通路活性的影響:共有11個(gè)差異基因富集到鈣離子通路中,硼主要調(diào)節(jié)鈣調(diào)磷酸酶(CaN)和鈣調(diào)素依賴蛋白激酶(CaMK)的活性。硼對(duì)CaN的兩個(gè)亞基均有調(diào)節(jié)作用,主要調(diào)控PPP3R1的活性,對(duì)PPP3CA的調(diào)節(jié)作用較小。硼能夠通過(guò)調(diào)節(jié)Ca N的活性,影響CaN下游轉(zhuǎn)錄因子NFAT和MEF2C的表達(dá)水平,參與Ca2+-CalcineurinNFAT信號(hào)通路活性調(diào)節(jié)。(3)硼對(duì)B細(xì)胞受體信號(hào)通路和T細(xì)胞受體信號(hào)通路活性的影響:共有7個(gè)差異基因富集到B細(xì)胞受體信號(hào)通路中,共有11個(gè)差異基因富集到T細(xì)胞受體信號(hào)通路中。T、B細(xì)胞受體能夠介導(dǎo)肌動(dòng)蛋白細(xì)胞骨架,MAPK通路,PI3K-AKT通路以及Ca N-NFAT通路的活化。根據(jù)KEGG和熒光定量分析結(jié)果,顯示硼能夠影響PI3K激酶的活性,調(diào)控PI3K-AKT通路。硼對(duì)T、B細(xì)胞因子受體通路的影響作用與MAPK通路,鈣離子通路以及PI3K-AKT通路的活性密切相關(guān)。(4)硼對(duì)Toll樣受體通路活性的影響:共有11個(gè)差異基因富集到Toll樣受體信號(hào)通路中,硼對(duì)Toll樣受體通路的調(diào)節(jié)包括兩部分,不僅能夠調(diào)控MyD88依賴性信號(hào)通路,也能夠調(diào)控MyD88非依賴性信號(hào)通路。并且高劑量硼對(duì)鴕鳥(niǎo)機(jī)體中Toll樣受體信號(hào)通路的調(diào)節(jié)呈現(xiàn)負(fù)調(diào)控調(diào)節(jié),抑制了Toll樣受體信號(hào)通路的活化,影響機(jī)體免疫應(yīng)答的機(jī)能。(5)硼對(duì)熱休克蛋白家族(HSP)的影響:熱休克蛋白與機(jī)體的細(xì)胞凋亡密切相關(guān),是一種抗凋亡蛋白。熱休克蛋白家族中的代表性成員是Hsp70,Hsp70的伴侶分子是Hsp40。高劑量的硼顯著抑制了Hsp70和Hsp40蛋白的表達(dá),抑制了鴕鳥(niǎo)機(jī)體內(nèi)熱休克蛋白的抗凋亡作用以及介導(dǎo)機(jī)體免疫應(yīng)答的能力,導(dǎo)致鴕鳥(niǎo)機(jī)體抵抗力降低。(6)硼對(duì)細(xì)胞凋亡通路及癌癥通路中相關(guān)基因的影響:硼主要影響TRAIL誘導(dǎo)的外源性細(xì)胞凋亡通路的活性,且參與凋亡抑制蛋白FLIP和IAP的活性調(diào)節(jié),對(duì)caspase家族的調(diào)節(jié)作用較小。硼參與鴕鳥(niǎo)機(jī)體內(nèi)癌癥信號(hào)通路的調(diào)節(jié),主要影響癌癥信號(hào)通路中Wnt信號(hào)通路,MAPK信號(hào)通路,PI3K-AKT信號(hào)通路以及細(xì)胞因子-受體互作通路的活性,這些信號(hào)通路發(fā)揮著復(fù)雜精細(xì)的調(diào)控作用,共同參與雛鴕鳥(niǎo)機(jī)體內(nèi)細(xì)胞的分化、增殖及凋亡過(guò)程。
[Abstract]:African ostrich has great economic value and scientific value. Ostrich rearing period is 3 months long. The artificial ostrich is susceptible to stress, infection and disease, which makes the ostrich morbidity and mortality rate as high as 10%-50%., the ostrich is fast growing bird, and the ostrich ostrich's individual development is different and immune function. The strength and weakness directly reflect the growth and development of ostrich in the brood stage. Thymus is an important central immune organ and lymphoid organ in the body. It is also an important immune organ for the body to resist external antigen stimulation and participate in the differentiation of T lymphocytes. Boron is a necessary trace element of the animal body and has many biological functions. Boron is used in the immune system. A moderate amount of boron is beneficial to the growth and development of immune organs. In this experiment, the 1 day old ostrich was selected as the research object, and a gradient dose of boric acid was added to the drinking water, and the thymus was killed at the age of 90 days and the thymus was slaughtered. The effect of Boron on the thymus of the ostrich central immune organ was studied to explain the immune response of boron to the ostrich body. The effect of.1. boron on the growth and development related genes of chick thymus Foxn1 participates in the differentiation and development of thymic epithelial cells, and plays a key regulatory role. BMP2 and BMP4 participate in the regulation of the development of thymus T cells. Foxn1, BMP2 and BMP4 genes are closely related to the homeostasis of the thymus environment. The effect of apoptosis and the regulation of the expression of Foxn1, BMP2 and BMP4 on the development of ostrich thymus development, to reveal the related mechanism of boron to the development of ostrich thymus development, provide reference.HE staining technique to observe the effect of Boron on the histological structure of the thymus of ostrich, RACE PCR clone ostrich Foxn1 gene sequence, immunohistochemical technique and Western blot Localization and expression of Foxn1 protein in the ostrich thymus by technique. The expression of BMP2 and BMP4 protein in the ostrich thymus was detected by immunofluorescence. The fluorescence quantitative PCR technique was used to detect the Foxn1 gene, BMP2 gene and the mRNA expression level of BMP4 gene to detect the apoptosis in the ostrich's thymus, and the immunohistochemical detection of activator caspase. The results were as follows: (1) the effects of Boron on the histological structure of the thymus of ostrich were observed as follows: (1) HE staining was used to observe the histological structure of the thymus. The morphological structure of the thymus in the high dose boron group (group B320 and B640) was incomplete, the boundary of the skin marrow junction was not clear, the appearance of "star empty" in the thymus cortex, the sharp decrease of thymus cells and the exhaustion of the thymus, and the thymus medulla In the B640 group, the thymus thymus structure was most serious, the thymus epithelial cell vacuolation and the medulla were the most obvious. The morphological structure of the thymus gland in the low dose boron group (B40 and B80) was the same as that in the control group. (2) the TUNEL technique was used to detect the apoptosis in the thymus of the ostrich, compared with the control group, the B80 and B160 groups were withered in the thymus gland. The number of dead cells was less, the number of apoptotic cells in the thymus of B320 and B640 increased and the apoptotic cells were distributed. (3) the detection of Caspase-3 activator found that the positive signal of Caspase-3 positive in the B320 and B640 groups was significantly enhanced. The high dose of boron induced the activation of Caspase-3 and induced the apoptosis of the ostrich thymocyte. (4) racepcr cloned and amplified ostrich. The bird Foxn1 gene amplified the conservative fragment of the ostrich Foxn1 gene with random primers. The length of the ostrich Foxn1 gene was 1477bp, the length of 5 'race and 3' race was 384bp and 1050bp, the whole length was 2736bp, and the 654 amino acids were encoded. The ostrich Foxn1 gene was highly conserved, and the homology of the Foxn1 genes of the Falcon, parrots, falcons, pigeons, ducks and chickens was 92.1, respectively. The positive signals of%, 91.1%, 90.8%, 89.6%, 88.1% and 83.5%. (5) Foxn1 were mainly distributed in the ostrich thymus medulla, and the thymic cortex had a small amount. The boric acid dose group that mainly labeled the thymic epithelial cell.80mg/l could significantly increase the expression of Foxn1 protein in the ostrich thymus, and the 640mg/l dose of boric acid could significantly reduce the Foxn1 protein in the thymus gland. (6) the expression level of mRNA in BMP2 and BMP4 showed a boron dose dependence and the expression trend increased first and then decreased. In the boron treatment group of 80mg/l, the expression of BMP2 and BMP4 was highly expressed in the ostrich thymus. In the boron treatment group of 640mg/l, the expression level of BMP2 and BMP4 was significantly suppressed. The addition of proper boron in drinking water to promote the hair of the chick thymus. The thymus development of ostrich is normal; excessive boron leads to the destruction of the histological structure of the thymus of ostrich, inducing apoptosis in the thymus of the ostrich, the expression of activator caspase-3 and the inhibition of the normal development of the thymus of the ostrich,.80mg/l boron promotes the expression of BMP2, BMP4, Foxn1 protein, BMP2, BMP4, and Foxn1 protein may increase the thymus epithelium fine. The differentiation and development of the cell promote the differentiation and development of T cells and enhance the immune resistance of the ostrich body. The high dose of boron (640mg/l) significantly inhibits the expression of BMP2, BMP4, Foxn1 protein in the ostrich thymus, resulting in the destruction and degradation of the ostrich thymus structure, the serious damage to the borderline junction of the thymus, the differentiation and development of the thymus epithelial cells, and the inhibition of the t. The normal growth and development of the cells, the reduction of the RNA-seq analysis of the thymus response to the thymus of ostrich.2. in order to study the mechanism of boron in the immune response of the ostrich, this study used RNA-seq technology to analyze the role of microelement boron in the ostrich thymus immune response for the first time. A total of 3 samples were selected for RNA-seq analysis, which were the control group ostrich thymus, B80 group ostrich thymus, and the b640 group ostrich thymus.Rna-seq analysis results showed: (1) 3 ostrich thymic sequence library (control sample library, B80 group library and b640 group sample library), including 32201730003427886800 and 3199872000 rawdata.c, respectively. The number of cleanreads in group ontrol, group B80 and b640 is 31753948003379919000 and 3157669200 respectively. The proportion of cleandata to reads is 98.61%, 98.60% and 98.68%. (2) control, respectively. The reads number of the ostrich reference genome of the B80 group and b640 group is 1192998912851275 and 1141161, respectively. 7, the proportion of total reads was 75.14%, 76.04% and 72.28%. (3) gene coverage statistical analysis showed that the proportion of gene number in the three library was 72%, 71% and 69%. (4), respectively: control and b640 group, B80 group and b640 group, control group and B80 group differential gene. The total total was 2044 (up - regulation 228, down gene 1816), 1085 (up - regulation 222, down - regulated gene 863) and 902 (up - regulation 309, down gene 593) differential gene. (5) difference gene trend analysis showed that all the difference genes were divided into 7 trends, trend 0, trend 1 Trend 3 has significant difference. Trend 0 contains 1290 differentially genes, and the expression in 3 libraries (control, B80 and b640) shows a trend of continuous decline; trend 1 contains 1030 differentially expressed genes, and the expression in 3 library is in turn decreasing and holding the same trend; trend 3 contains 1487 differentially genes, The expression in the 3 libraries showed a trend of first or later decline. (6) the analysis of the enrichment analysis of the difference gene KEGG signaling pathway showed that the cytokine receptor interaction pathway, the cancer pathway, the calcium ion signaling pathway, the actin cytoskeleton regulation pathway and the MAPK signaling pathway were enriched in the 10 signal pathways. The signal pathway mainly participates in the immune response and inflammatory response process of the body. (7) the results of the KEGG enrichment of differential genes in the paired samples show that boron also participates in the toll like receptor signaling pathway, the B cell receptor pathway, the T cell receptor pathway and the apoptosis pathway. The effects of related signaling pathways include MAPK signaling pathway, calcium channel, B cell receptor pathway, T cell receptor pathway, actin regulation signaling pathway, toll like receptor signaling pathway, apoptosis signaling pathway, and cancer signal pathway.3. boron on ostrich body immunity, inflammation and growth related pathways in order to further examine The results of RNA-seq analysis, and the effect of different doses of Boron on the immune function related signaling pathway in the ostrich's thymus, were studied in this project, including the MAPK pathway, calcium channel, B cell receptor pathway, T cell receptor pathway, toll like receptor pathway, and cancer signaling pathway. Road and apoptotic pathway. (1) the effect of Boron on the activity of MAPK pathway: a total of 27 different genes were enriched in the MAPK signaling pathway, most of the gene expression in group control and B80 increased, while most of the genes in the b640 group were down regulated, 24 of the 27 genes decreased and 3 genes increased. Boron could affect ostrich. The ras/erk pathway, the JNK pathway and the p38MAPK pathway in the bird's body, but not involved in the regulation of the ERK5 pathway,.Qrt-pcr and Westernblot verification showed that 80mg/l boron promoted the expression of p-ERK, p-JNK and p-p38 protein levels in the ostrich thymus. With the increase of boron dosage, p-ERK, p-JNK and protein expression level gradually decreased, in the 640 boron histone. The lowest expression level. Boron can regulate the expression level of kinase in ERK, JNK and p38MAPK pathways and influence the activity of MAPK signaling pathway. (2) the effect of Boron on the activity of calcium ion pathway: a total of 11 different genes are enriched in the calcium channel, boron mainly regulates the activity of calcineurin (CaN) and calmodulin dependent protein kinase (CaMK). Boron is to CaN The two subunits can regulate the activity of PPP3R1 and regulate the activity of PPP3CA. Boron can regulate the expression level of NFAT and MEF2C by regulating the activity of Ca N, and participate in the activity regulation of Ca2+-CalcineurinNFAT signaling pathway. (3) the boron on the B cell receptor signaling pathway and the T cell receptor signaling pathway. A total of 7 differential genes are enriched in the B cell receptor signaling pathway, and a total of 11 different genes are enriched into the T cell receptor signaling pathway.T, and the B cell receptor can mediate the actin cytoskeleton, the MAPK pathway, the PI3K-AKT pathway and the activation of the Ca N-NFAT pathway. The activity of PI3K kinase regulates the activity of the PI3K-AKT pathway. The effect of Boron on the T, B cell factor receptor pathway is closely related to the MAPK pathway, the calcium channel and the activity of the PI3K-AKT pathway. (4) the effect of Boron on the activity of Toll like receptor pathway: a total of 11 different genes are enriched in the Toll like receptor signaling pathway, and the modulation of boron to the Toll like receptor pathway The two part includes not only the regulation of the MyD88 dependent signaling pathway but also the regulation of the MyD88 non dependent signaling pathway, and the regulation of the Toll like receptor signaling pathway in the ostrich body is negatively regulated by high dose boron, which inhibits the activation of the Toll like receptor signaling pathway and affects the function of the immune response. (5) boron to heat shock. The influence of protein family (HSP): the heat shock protein is closely related to the apoptosis of the body and is an anti apoptotic protein. The representative member of the heat shock protein family is Hsp70. The Hsp70 partner is the high dose of Hsp40., which inhibits the expression of Hsp70 and Hsp40 protein, and inhibits the anti apoptosis of the heat shock protein in the ostrich body. The ability to use and mediate the immune response of the body leads to the reduction of the resistance of the ostrich body. (6) the effect of Boron on the apoptosis pathway and the related genes in the cancer pathway: Boron mainly affects the activity of the exogenous apoptosis pathway induced by TRAIL, and is involved in the regulation of the activity of FLIP and IAP, and the regulation of the caspase family is more effective. Boron is involved in the regulation of the cancer signal pathway in the ostrich body, which mainly affects the activity of Wnt signaling pathway, MAPK signaling pathway, PI3K-AKT signaling pathway and cytokine receptor interaction pathway in the cancer signaling pathway. These signaling pathways play a complex and fine regulatory role, and participate in the differentiation, proliferation and proliferation of cells in the ostrich body. The process of apoptosis.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S839
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本文編號(hào)：1872760