基因组学与应用生物学
基因組學與應用生物學
기인조학여응용생물학
Genomics and Applied Biology
2011年
4期
257-264
,共8页
朱友银%田晓娥%赵德刚
硃友銀%田曉娥%趙德剛
주우은%전효아%조덕강
融合筛选标记%Bar%gus%融合重组酶位点%LoxP/FRT%植物遗传转化
融閤篩選標記%Bar%gus%融閤重組酶位點%LoxP/FRT%植物遺傳轉化
융합사선표기%Bar%gus%융합중조매위점%LoxP/FRT%식물유전전화
Fused selectable marker gene%Bar：：gus%Fused recombinase site%LoxP/FRT%Plant genetic transformation
本研究利用化学合成法合成了包含pCAMBIA0390左右边界T-DNA和LoxP／FRT（LF）位点的DNA片段Ⅰ，利用SacⅡ和SphⅠ酶切位点，去除了pCAMBIA0390左右边界T—DNA和多克隆位点之间的序列，然后连接DNA片段Ⅰ和载体片段，构建了植物表达载体pGM323-LF-enTP。随后，再合成含有适合在单予叶植物中表达的由玉米Ubi-1启动子驱动的融合标记基因（Bar：：gus）和水稻actin-1启动子驱动的助抗虫蛋白基因（Cry1A6）表达元件的DNA片段Ⅳ，在pGM323～LF—enTP的基础上，利用5以Ⅰ和PstⅠ位点构建了同时含有LF位点、Bar：：gus以及Cry1Ab基因表达元件的表达载体pGM626-LF—ABt。利用含有pGM626-LF—ABt的农杆菌遗传转化烟草和玉米，以草丁膦作为抗性筛选剂，非转化细胞得到了有效抑制，快速获得了转基W植株，利用GUS组织化学检测和RT—PCR分析了转基因植株中标记基因的表达，结果表明pGM626-LF—ABt可以用于农杆菌介导的单、双子叶植物遗传转化。本研究为培育安全抗虫转基因植物奠定了基础。
本研究利用化學閤成法閤成瞭包含pCAMBIA0390左右邊界T-DNA和LoxP／FRT（LF）位點的DNA片段Ⅰ，利用SacⅡ和SphⅠ酶切位點，去除瞭pCAMBIA0390左右邊界T—DNA和多剋隆位點之間的序列，然後連接DNA片段Ⅰ和載體片段，構建瞭植物錶達載體pGM323-LF-enTP。隨後，再閤成含有適閤在單予葉植物中錶達的由玉米Ubi-1啟動子驅動的融閤標記基因（Bar：：gus）和水稻actin-1啟動子驅動的助抗蟲蛋白基因（Cry1A6）錶達元件的DNA片段Ⅳ，在pGM323～LF—enTP的基礎上，利用5以Ⅰ和PstⅠ位點構建瞭同時含有LF位點、Bar：：gus以及Cry1Ab基因錶達元件的錶達載體pGM626-LF—ABt。利用含有pGM626-LF—ABt的農桿菌遺傳轉化煙草和玉米，以草丁膦作為抗性篩選劑，非轉化細胞得到瞭有效抑製，快速穫得瞭轉基W植株，利用GUS組織化學檢測和RT—PCR分析瞭轉基因植株中標記基因的錶達，結果錶明pGM626-LF—ABt可以用于農桿菌介導的單、雙子葉植物遺傳轉化。本研究為培育安全抗蟲轉基因植物奠定瞭基礎。
본연구이용화학합성법합성료포함pCAMBIA0390좌우변계T-DNA화LoxP／FRT（LF）위점적DNA편단Ⅰ，이용SacⅡ화SphⅠ매절위점，거제료pCAMBIA0390좌우변계T—DNA화다극륭위점지간적서렬，연후련접DNA편단Ⅰ화재체편단，구건료식물표체재체pGM323-LF-enTP。수후，재합성함유괄합재단여협식물중표체적유옥미Ubi-1계동자구동적융합표기기인（Bar：：gus）화수도actin-1계동자구동적조항충단백기인（Cry1A6）표체원건적DNA편단Ⅳ，재pGM323～LF—enTP적기출상，이용5이Ⅰ화PstⅠ위점구건료동시함유LF위점、Bar：：gus이급Cry1Ab기인표체원건적표체재체pGM626-LF—ABt。이용함유pGM626-LF—ABt적농간균유전전화연초화옥미，이초정련작위항성사선제，비전화세포득도료유효억제，쾌속획득료전기W식주，이용GUS조직화학검측화RT—PCR분석료전기인식주중표기기인적표체，결과표명pGM626-LF—ABt가이용우농간균개도적단、쌍자협식물유전전화。본연구위배육안전항충전기인식물전정료기출。
DNA fragment Ⅰ containing T-DNA ofpCAMBIA0390 vector and LoxP/FRT （LF） sites were synthesized with additional Sac＇ Ⅱ and Sph Ⅰ recognition sites. Then the vector was released from pCAMBIA0390, and the sequences between T-DNA and multiple cloning sites of T-DNA of pCAMBIA0390 vector were deleted with restriction enzyme Sac Ⅱ and Sph Ⅰ . Digested sequences were ligated to generate pGM323-LF-enTP plasmid. DNA fragment Ⅳ was synthesized with the fused Bar：：gus gene under maize ubiquitin promoter （Ubi- 1） and CrylA b under the rice actin-1 gene （Act-1） promoter. The vector containing LF and T-DNA sequences was released from pGM323-LF-enTP plasmid with Sa/I and Pst I sites. Then digested sequences were ligated to generate pGM626- LF-ABt plasmid. Transformed explants were cultured in selection medium containing bialaphos. Putatively transformed events were covered after selection cultivation. The expression of fused selectable marker gene was analyzed in different tissues of transgenic tobacco and transgenic maize. The results confirmed that the plasmid pGM626-LF-ABt could be used for plant transformation. These results would be a base for breeding transgenic plants with bio-safety.
