本文選題：GFP + 高保真酶�。� 參考：《西南農(nóng)業(yè)學(xué)報(bào)》2017年09期

【摘要】：【目的】為利用綠色熒光蛋白基因(GFP)的表達(dá)情況優(yōu)化聚乙二醇(PEG)介導(dǎo)的小立碗蘚原生質(zhì)體的轉(zhuǎn)化體系及檢測CAD1基因在細(xì)胞中的定位,構(gòu)建小立碗蘚GFP-CAD1-PTN182融合基因表達(dá)載體。【方法】用高保真酶獲得GFP基因,通過雙酶切GFP基因和CAD1-PTN182表達(dá)載體,將兩片段洗脫回收,然后用T4-DNA連接酶將酶切產(chǎn)物在16℃條件下過夜連接。將連接產(chǎn)物轉(zhuǎn)化到E.coli DH5α感受態(tài)細(xì)胞中,通過凝膠成像電泳的方法,挑選陽性克隆進(jìn)行驗(yàn)證,并經(jīng)過酶切驗(yàn)證和測序�！窘Y(jié)果】序列對比相似度達(dá)100%,表明GFP-CAD1-PTN182融合基因表達(dá)載體構(gòu)建成功�！窘Y(jié)論】該載體的成功構(gòu)建,為PEG介導(dǎo)小立碗蘚原生質(zhì)體轉(zhuǎn)化體系的優(yōu)化提供了便捷的檢測手段,同時(shí)為進(jìn)一步研究CAD1基因在細(xì)胞中的定位提供了理論依據(jù)。
[Abstract]:[objective] to optimize the transformation system of PEG- mediated protoplasts and to detect the location of CAD1 gene in cells by using the expression of GFP gene. The expression vector of GFP-CAD1-PTN182 fusion gene was constructed. [methods] GFP gene was obtained by high fidelity enzyme. The two fragments were eluted and recovered by double enzyme digestion of GFP gene and CAD1-PTN182 expression vector. The digested products were linked overnight at 16 鈩，

本文編號：1884711