Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22165
標題: Studies on the Molecular Genetic Markers Associated with Reproduction of Ducks Using DNA Microarray
利用微陣列基因晶片探討鴨繁殖性狀之分子遺傳標記
作者: 黃秀琳
Huang, Hsiu-Lin
關鍵字: duck;鴨;DNA microarray;DNA polymorphisms;Genotyping, Marker-assisted selection;DNA晶片;DNA多態性;基因型鑑定;標記輔助選拔
出版社: 分子生物學研究所
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摘要: 
DNA microarray technology was used for the studies on differentially-expressed genes in oviduct of Tsaiya ducks in Taiwan. The genotypes associated with reproductive performance for marker-assisted selection were also investigated. The magnum and utero-vaginal junction (UV-J) in oviduct were employed for the hatchability and the fertile-period experiments, respectively. In the hatchability study, transcriptome analysis using home-made cDNA microarray was performed to identify differentially-expressed genes that are correlated with hatchability, and a new PCR-RFLP marker of high hatchability among the identified genes was observed. The cDNA microarray technique was used for gene expression profiling of the magnum epithelium of laying Tsaiya ducks, and several regulated genes associated with hatchability were found. The results of real-time PCR and Western blotting analysis confirmed that the mRNA and protein levels of ovomucoid in the magnum epithelium of animals in the low-hatchability group were significantly higher than the levels in the high-hatchability group (p<0.05). Primers TovF1and TovR1 designed according to the ovomucoid EST sequence were used to amplify genomic DNA samples of different individual Tsaiya ducks, and sequence analysis of the amplified DNA products showed deletion among the ducks from the low-hatchability group. Primers TovF2 andTovR2 then probed inside the amplified DNA products and were used to perform PCR-RFLP analysis to classify the ducks into +/+, +/- and -/- genotypes. The animals of +/+ and +/- genotypes were identified to have a significantly higher hatchability than those of the -/- genotype (p<0.05). In contrast, no differences were observed between genotypes in terms of fertility, fertile period, egg weight or total number of eggs. The results indicated that a novel PCR-RFLP marker of high hatchability, the ovomucoid gene polymorphisms, can be used as a genetic marker for marker-assisted selection to improve hatchability in Tsaiya ducks. In the fertile-period study, prolonged fertile period increases the efficiency of fertilization. Here, RNA was extracted from long- and short-fertile-period groups, converted to double cDNA which was then in vitro transcripted to complementary RNA (cRNA). Affymetrix chips were hybridized with these cRNA, three biological repeats were performed, and 27 transcripts were identified as being differentially-expressed. Interestingly, by searching the annotated pathway databases, the results demonstrated that Neuropeptide Y (NPY), the RNA expression of which was increased by 2.96-fold in the short-fertile-period group as compared with the long-fertile-period group in the experiment described in this thesis, has been shown to reduce blood flow and substance supply to local tissues. Enah/Vasp-like (EVL), the RNA expression of which was significantly increased by 1.77-fold in the short-fertile-period group as compared with the long-period group, has been demonstrated to be important in activated T-cells. In contrast, trafficking kinesin-binding protein 1 (TRAK1), the expression of which was increased by 2.33-fold in the long-period group as compared with the counterparts, has been suggested to inhibit precocious activation of sperm and prolong sperm life in female sperm reservoir.

本研究之目的在利用微陣列基因晶片探討鴨輸卵管之蛋白分泌部及宮道接合組織差異表現的基因,進一步研究其與孵化率及受精持續天數等繁殖性狀有關基因型的相關性,期能供標記輔助選拔之用。在孵化率的試驗中,利用自行點製的cDNA晶片找尋與此性狀相關之差異表現基因,並在這些基因中,找到了新的RFLP-PCR標記,可用來選拔高孵化率個體: cDNA晶片的數據顯示,在低孵化率群中,ovomucoid之mRNA表現量大於高孵化率族群者1.5倍,real-time PCR的數據與來自晶片的結果相符。我們也比較ovomucoid蛋白質表現量在高、低孵化率兩族群間的差異,結果顯示,低孵化率族群的ovomucoid蛋白比高孵化率群的表現量高(p<0.05)。以ovomucoid EST序列設計TovF1及TovR1引子,此對引子可以成功由基因組核苷酸序列增殖出ovomucoid片段,因此得以發現在ovomucoid基因組核苷酸序列中的多態性,即在低孵化率族群中出現具有核苷酸缺失的片段,相對的,完整核苷酸片段出現在高孵化率族群。進一步以TovF2及TovR2引子利用PCR-RFLP可以鑑定出+/+、+/-及-/-三種多態性,且發現-/-基因型與低孵化率有顯著相關,而+/+及+/-型者有較高的孵化率(p<0.05)。這三種基因型則與其他重要的繁殖性狀:受精持續天數、受精率、蛋重、蛋數無關。綜合以上孵化率的試驗得知,ovomucoid基因組核苷酸序列中的多態性可作為新的RFLP-PCR標記及輔助選拔的依據。在受精持續天數的試驗中,因為精子進入母體的生殖器官後,精子會先被儲存起來,直到卵被排出後,精子由儲存的地方逐漸被釋出。如此,除了可以經由精子與母體宮道接合處的接觸維持精子壽命,也能在適當的時間使精子與卵結合,增加受精率。本試驗分別由長及短受精持續天數的鴨宮道接合處萃取RNA,轉成雙股cDNA,再進行體外轉錄合成cRNA,而後與Affymetix的晶片作雜交,共完成三次獨立重複試驗,來尋找母體儲精處中幫助或抑制延長精子停留時間及壽命的基因。值得注意的是,將Affymetix晶片結果與生理路徑資料庫及文獻做比對之後,我們篩選出下列可能在決定受精持續天數有意義的基因,進一步討論其可能的機制,且再次確認Affymetix晶片結果:可能造成血管與組織間缺血的Neuropeptide Y (NPY) 其mRNA表現量在短受精持續天數組中顯著上升2.96倍,相對於長受精持續天數組。可能顯示對外來精子有過敏反應的Enah/Vasp-like (EVL) 其mRNA表現量在短受精持續天數組中也顯著上升1.77倍。當相較於短受精持續天數組時,可能抑制精子不正常活化的TRAK1其mRNA表現量在長受精持續天數組中有2.33倍的顯著上升。此外,試驗結果亦顯示,real-time PCR與Affymetix晶片所得基因之表現狀況一致。因此TRAK1、EVL、NPY表現量的不同可能改變受精持續天數。
URI: http://hdl.handle.net/11455/22165
其他識別: U0005-2101201113355800
Appears in Collections:分子生物學研究所

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