广西植物
廣西植物
엄서식물
GUIHAIA
2013年
1期
76-81
,共6页
水稻根对敏感基因 2.1%猎物载体%酵母双杂交%自激活%毒性检测
水稻根對敏感基因 2.1%獵物載體%酵母雙雜交%自激活%毒性檢測
수도근대민감기인 2.1%작물재체%효모쌍잡교%자격활%독성검측
rice ROOT UV‐B SENSITIVE 2 .1(OsRUS2 .1)%prey vector%yeast two hybrid%self‐activated transcrip‐tional activity%toxicity detection
采用 PCR 技术扩增水稻根 UV‐B 敏感基因2.1(ROOT UV‐B S EN S IT IV E 2.1,RUS2.1)四个不同片段[OsRUS2.1(1‐1317),OsRUS2.1(1‐138),OsRUS2.1(139‐879),OsRUS2.1(880‐1317)],连接到 T 载体pMD18‐T‐Simple 上,测序无误后分别亚克隆到猎物表达载体 pGADT7上.结果表明:四个 OsRUS2.1基因片段的表达载体构建成功,读码框正确;分别转化这四个猎物表达载体于酵母感受态细胞 Y187中,用 LacZ 、M EL1活性检测法和营养缺陷型培养基 SD‐Leu‐DO 培养法进行自激活检测和毒性检测,结果表明构建的四个 OsRUS2.1不同片段的猎物表达载体对酵母菌株 Y187均没有转录激活活性和毒害作用,可用于后续研究.
採用 PCR 技術擴增水稻根 UV‐B 敏感基因2.1(ROOT UV‐B S EN S IT IV E 2.1,RUS2.1)四箇不同片段[OsRUS2.1(1‐1317),OsRUS2.1(1‐138),OsRUS2.1(139‐879),OsRUS2.1(880‐1317)],連接到 T 載體pMD18‐T‐Simple 上,測序無誤後分彆亞剋隆到獵物錶達載體 pGADT7上.結果錶明:四箇 OsRUS2.1基因片段的錶達載體構建成功,讀碼框正確;分彆轉化這四箇獵物錶達載體于酵母感受態細胞 Y187中,用 LacZ 、M EL1活性檢測法和營養缺陷型培養基 SD‐Leu‐DO 培養法進行自激活檢測和毒性檢測,結果錶明構建的四箇 OsRUS2.1不同片段的獵物錶達載體對酵母菌株 Y187均沒有轉錄激活活性和毒害作用,可用于後續研究.
채용 PCR 기술확증수도근 UV‐B 민감기인2.1(ROOT UV‐B S EN S IT IV E 2.1,RUS2.1)사개불동편단[OsRUS2.1(1‐1317),OsRUS2.1(1‐138),OsRUS2.1(139‐879),OsRUS2.1(880‐1317)],련접도 T 재체pMD18‐T‐Simple 상,측서무오후분별아극륭도작물표체재체 pGADT7상.결과표명:사개 OsRUS2.1기인편단적표체재체구건성공,독마광정학;분별전화저사개작물표체재체우효모감수태세포 Y187중,용 LacZ 、M EL1활성검측법화영양결함형배양기 SD‐Leu‐DO 배양법진행자격활검측화독성검측,결과표명구건적사개 OsRUS2.1불동편단적작물표체재체대효모균주 Y187균몰유전록격활활성화독해작용,가용우후속연구.
@@@@Four fragments of rice(Oryza sativa)ROOT UV‐B SENSIT IV E 2 .1(OsRUS2 .1) ,OsRUS2 .1(1‐1317) , OsRUS2 .1(1‐138) ,OsRUS2 .1 (139‐879) ,OsRUS2 .1 (880‐1317) ,were amplified by PCR from cloned OsRUS2 .1 plasmid ,and were ligated with pMD18‐T‐simple vector ,then transformed to E .coli TOP10 competent cell .The posi‐tive clones were selected and sequenced .The confirmed fragments were subcloned to prey vector pGADT 7 .The four constructed pGADT7 prey vectors were further confirmed by enzyme digestion and sequencing . The confirmed 4 types of pGADT7 prey vectors were transformed to Y187 yeast competent cells .The self‐activation and toxicity of the plasmids to host yeast Y187 were detected by LacZ and MEL1 activity assays and culturing in auxotroph medium SD‐Leu ‐DO .Results showed that the four constructed plasmids had no self‐transcriptional activity and not toxicity to yeast strain Y187 ,and they could be used in the following yeast two‐hybrid experiments .
