臺灣博碩士論文加值系統

水產養殖是台灣相當重要的產業，佔台灣漁業總產值的35%，而飼料成本約占總成本的40%，所以魚粉的價格影響到養殖漁業的穩定收益。魚粉的產量低及價格的變動不穩定，大豆的價格便宜及供應平穩，適合做為取代魚粉的原料。大豆種子富含有蛋白質及平衡的胺基酸組成分，其中β-conglycinin蛋白質含量占種子總蛋白的35%。大豆蛋白與魚粉相較之下，缺乏胺基酸methionine及lysine，以及添加glycine可以改善大豆適口性。本研究即是分別把含有六套MGKMGR及十套GMKGMR之重覆套數的核酸片段插入大豆β-伴球蛋白(β-conglycinin)之β-亞基 (β-subunit) (7Sb)基因，利用大豆作為生物反應器生產優質水產飼料用蛋白。
本研究將二個改造之β-亞基基因 (7Sb-M56及7Sb-G510)自E. coli 表現載體pET21-7Sb-G510及pET21-7Sb-M56上切下，再黏接到含有CaMV35S 啟動子(持續性表現) 及GmPM9啟動子(種子專一表現) 的植物基因轉殖載體 pCambia 1304。轉殖載體以mgfp及gusA為報導基因，以hptII作為篩選基因，總共完成構築四種植物轉殖載體：pM56-1304-gus、pG510-1304-gus、pGm9-M56-1304-gus、 pGm9-G510-1304-gus。
將四種植物轉殖載體利用農桿菌基因轉殖法轉殖到''高雄選10號''大豆子葉節。培殖體以100 mg/L的hygromycin進行篩選，並誘導再生。再生植株及T1後裔均經由PCR、RT-PCR、西方墨點等方法檢測。目前已確認12個T1轉殖系帶有7Sb-G510基因，並表現 7Sb-G510 mRNA及7Sb-G510 蛋白。

Aquaculture contributes around 35% of the value of the Taiwan’s total seafood production, and is an important sector of fishery industry. Feed cost accounts 40% of the total production cost. Stable income of aquaculture is dependent on the price of fish meal. However, fish meal is in short supply and expensive, it is necessary to develop new product to substitute for the fish meal in aqua feeds. The high protein content, balanced amino acid composition, steady supply, low cost of soybean made the derived products to be the ideal ingredients to replace the fish meal. The intrinsic limitations associated with soybean seed protein are low methionine content and poor palatability for fish. Attempts had been made to modify the soybean β-subunit of β-conglycinin gene (7Sb) by inserting the nucleotides of six and ten repeats of MGKMGR (7Sb-M56) and GMKGMR (7Sb-G510), respectively, so as to modify the amino acid composition of the soybean protein as a remedy.
The modified 7Sb genes driven by the CaMV35S or Gm9 promoter (soybean seed-specific) had been sub-cloned onto pCambia 1304 vector which is anticipated to provide with stable supply of aquaculture meal thus benefiting farmers with better and stable income. Five plasmids had been constructed for soybean transformation, which were named pM56-1304-gus, pG510-1304-gus pGm9-M56-1304-gus, and pGm9- G510-1304-gus.
These plasmids were used for soybean transformation. Cotyledon nodes were used as explants for transformation of soybean [Glycine max (L.) Merr cv. ''Kaohsiung 10''] by Agrobacterium-mediated method. The transformed explants were selected with 100 ppm hygromycin. The regenerated plants and T1 progeny were examined by PCR, RT-PCR, and western blots. Twelve T1 progenies of 7Sb-G510 transgenic soybean were confirmed as evidenced by the presences of 7Sb-G510 gene, 7Sb-G510 mRNA, and 7Sb-G510 protein.

中文摘要………………………………………………………………… 1
英文摘要………………………………………………………………… 2
前言……………………………………………………………………… 3
前人研究………………………………………………………………… 5
一、植物作為生物反應器……………………………………………… 5
二、大豆儲存性蛋白－11S球蛋白(glycinin)與7S
β-伴球蛋白(β-conglycinin)……………………………………… 7
三、大豆粉取代魚粉作為水產飼料用蛋白…………………………… 9
四、大豆農桿菌基因轉殖…………………………………………… 10
五、大豆培殖體再生………………………………………………… 12
材料與方法………………………………………………………………13
結果…………………………………………………………………… 22
一、pET21-7Sb-M56及pET21-7Sb-G510載體之檢驗與蛋白
表現分析………………………………………………………… 22
二、pM56-1304-gus、pG510-1304-gus、pGm9-M56-1304-gus、
pGm9-G510- 1304-gus轉殖載體之構築………………………… 23
三、大豆農桿菌基因轉殖之培殖體篩選及誘導植株再生………… 25
四、大豆轉殖植株之之基因及表現分析…………………………… 25
五、轉殖大豆種子之抗hygromycin篩選與轉殖
後裔植株(T1)之基因及表現分析……………………………… 26
討論…………………………………………………………………… 53
參考文獻…………………………………………………………… 57
附錄…………………………………………………………………… 65

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