褐色菜鴨殼腺黏膜細胞中與蛋殼形成相關基因之表現與選殖

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Title:	褐色菜鴨殼腺黏膜細胞中與蛋殼形成相關基因之表現與選殖
Other Titles:	Expression and molecular cloning of eggshell formation related genes in brown Tsaiya duck eggshell gland
Authors:	呂建和 Liu, Jian-He
Contributors:	歐柏榮 Ou, Bor-Rung 東海大學畜產與生物科技學系
Keywords:	褐色菜鴨;殼腺;蛋殼形成 brown Tsaiya duck;eggshell gland;eggshell formation
Date:	2002
Issue Date:	2011-05-19T05:48:21Z (UTC)
Abstract:	本實驗主要目的是在研究菜鴨蛋殼形成相關的基因，根據前人的研究，實驗著重於影響形成外殼膜、內殼膜的lysyl oxidase基因；運送鈣至蛋殼腺分泌部以影響蛋殼行鈣化作用的calbindin D28K基因；及一個由DDRT-PCR所篩選之未知基因(5-A-1)。藉由菜鴨殼腺黏膜RNA之萃取及RT-PCR擴增反應後，確定菜鴨殼腺黏膜細胞有這三個基因的表現；將所得的PCR產物定序及分析，發現實驗所得基因為菜鴨之lysyl oxidase及calbindin D28K基因，但5-A-1基因在GenBank基因庫中尚無發現相似之基因，故對其作用一無所知。另外，在分析此三個基因在不同排卵時期的表現量的實驗中，發現lysyl oxidase基因在剛排卵時的表現量最高；calbindin D28K基因在排卵後18小時有最高的表現量；5-A-1基因則在排卵後至6小時的表現量最高。為了進一步探討實驗所得基因之表現是否與蛋殼品質有關，實驗萃取強蛋殼品系與弱蛋殼品系之菜鴨殼腺黏膜細胞之RNA，分別加入lysyl oxidase 、calbindin D28K及5-A-1基因之引子，進行RT-PCR擴增反應，結果發現此三個基因在強蛋殼品系表現量比弱蛋殼品系表現量要來的高。為了進一步分析菜鴨calbindin D28K基因之可能作用，將所得之cDNA進行DNA定序及分析，結果發現菜鴨calbindin D28K基因之coding region為789 bp, 共具有四個calcium-binding damain、一個CK2 phosphorylation site、兩個protein kinase C phosphorylation site及一個Tyrosine phosphorylation site。由以上結果可知，本實驗所研究的lysyl oxidase、calbindin D28K及5-A-1基因之表現與蛋殼形成有關；另外，菜鴨calbindin D28K基因由於有EF-hand 之結構，推測此基因與鈣離子結合的特性可能和鈣離子的吸收與輸送有密切關係。 The goal of this study was to investigate the genes expressed during Tsaiya duck eggshell calcification / formation. We focused on the following three genes : the lysyl oxidase gene encoding a polypeptide that is important during inner and outer-membranes formation, the calbindin D28K gene encoding a product that transports calcium to the eggshell gland and the 5-A-1 gene, a novel gene amplified by DDRT-PCR from eggshell gland. Three PCR products were obtained from Tsaiya duck eggshell gland and these sequences were subsequently analyzed. The sequence of PCR products corresponding to the lysyl oxidase and calbindin D28K genes are very similar to these of chick, howrver, no match was found for the sequence 5-A-1 gene. Additionally, results from RT-PCR analysis indicated that the maximal levels of gene expression for lysyl oxidase, calbindin D28K, and 5-A-1 during different oviposition stages were found at 0, 18 and 6 hr immediately after oviposition, respectively. To investigate the involvement of these genes in eggshell quality, we examined the expression of these genes in eggshell glands of Tasiya ducks with strong and weak eggshell strength. The results showed that the expression of these 3 genes were higher in strong eggshell- strength Tsaiya duck than in those weak eggshell- strength one. To further analyzed the functions of calbindin D28K gene in Tsaiya duck, we analyzed DNA sequence of calbindin D28K by Omaga computer program. According to DNA sequence, we found the coding region of calbindin D28K gene in Tsaiya duck is 789 bp, and contains four calcium binding domains, one CK2 phosphorylation site, two PKC phosphorylation sites and one TYR phosphorylation site. In conclusion, the expressions of lysyl oxidase, calbindin D28K, and 5-A-1 genes were involved in eggshell formation. The DNA sequence of calbindin D28K gene containing the EF-hand structure suggests that the calcium-binding characteristic of this gene is highly relative with eggshell quality.
Appears in Collections:	[畜產與生物科技學系所] 碩博士論文

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