中国蔬菜
中國蔬菜
중국소채
CHINA VEGETABLES
2013年
20期
24-31
,共8页
杨朴丽%许俊强%刘智宇%王志敏%张丹华%汤青林%宋明
楊樸麗%許俊彊%劉智宇%王誌敏%張丹華%湯青林%宋明
양박려%허준강%류지우%왕지민%장단화%탕청림%송명
结球甘蓝%雌蕊%SPT%基因克隆%序列分析
結毬甘藍%雌蕊%SPT%基因剋隆%序列分析
결구감람%자예%SPT%기인극륭%서렬분석
Cabbage%Pistil%SPT%Moclecular cloning%Sequence analysis
以结球甘蓝E1为材料,提取花蕾总RNA,反转录cDNA。根据拟南芥SPT基因设计引物,采用同源克隆的方法从中克隆SPT基因序列1085 bp,开放阅读框1062 bp。通过cDNA推导得到的氨基酸序列分析表明,BoSPT编码353个氨基酸残基,预测分子量为37.67 kD,pI为6.83。经过EcoRⅠ和KpnⅠ限制酶双酶切后,构建原核表达质粒pET43.1a-BoSPT转化表达菌株E. coli Rosetta(DE3),通过SDS-PAGE 检测该蛋白的表达。经Smart-embl预测其具有bHLH家族结构域,位于序列第173~221位氨基酸残基处。进化树表明结球甘蓝BoSPT与拟南芥AtSPT和筷子芥AlSPT的亲缘关系较近。BoSPT基因的原核表达得到纯化的融合蛋白。
以結毬甘藍E1為材料,提取花蕾總RNA,反轉錄cDNA。根據擬南芥SPT基因設計引物,採用同源剋隆的方法從中剋隆SPT基因序列1085 bp,開放閱讀框1062 bp。通過cDNA推導得到的氨基痠序列分析錶明,BoSPT編碼353箇氨基痠殘基,預測分子量為37.67 kD,pI為6.83。經過EcoRⅠ和KpnⅠ限製酶雙酶切後,構建原覈錶達質粒pET43.1a-BoSPT轉化錶達菌株E. coli Rosetta(DE3),通過SDS-PAGE 檢測該蛋白的錶達。經Smart-embl預測其具有bHLH傢族結構域,位于序列第173~221位氨基痠殘基處。進化樹錶明結毬甘藍BoSPT與擬南芥AtSPT和筷子芥AlSPT的親緣關繫較近。BoSPT基因的原覈錶達得到純化的融閤蛋白。
이결구감람E1위재료,제취화뢰총RNA,반전록cDNA。근거의남개SPT기인설계인물,채용동원극륭적방법종중극륭SPT기인서렬1085 bp,개방열독광1062 bp。통과cDNA추도득도적안기산서렬분석표명,BoSPT편마353개안기산잔기,예측분자량위37.67 kD,pI위6.83。경과EcoRⅠ화KpnⅠ한제매쌍매절후,구건원핵표체질립pET43.1a-BoSPT전화표체균주E. coli Rosetta(DE3),통과SDS-PAGE 검측해단백적표체。경Smart-embl예측기구유bHLH가족결구역,위우서렬제173~221위안기산잔기처。진화수표명결구감람BoSPT여의남개AtSPT화쾌자개AlSPT적친연관계교근。BoSPT기인적원핵표체득도순화적융합단백。
Taking cabbage(Brassica oleracea L.)E1 as materials,We extracted total RNA from capullo,reverse transcribed cDNA. According to SPT gene sequence of Arabidopsis,primers were designed and 1 085 bp SPT gene with 1 062 bp open reading frame(ORF)was cloned by ho-mology cloning techniques. The deduced BoSPT protein contained 353 amino acids,with a molecu-lar weight of 37.67 kD and pI of 6.83. After double enzyme of EcoRI and KpnI restriction enzymes, and then construct the recombinant plasmids pET43.1a-BoSPT. After transformation to E. coli Ro-setta(DE3),the expression of recombinant proteins were detected via SDS-PAGE. The structural analysis of BoSPT though Smart-embl showed that it contained bHLH family domain,which located at the position of 173-221 amino acid residues,The phylogenetic tree indicated that the BoSPT had close genetic relationship with AtSPT and AlSPT. Prokaryotic expression showed that the molecular mass of BoSPT protein was purified.