CAJ | 학술논문

文章提取拟南芥植株总RNA ，通过RT-PCR扩增出AtMYB50与AtMYB61基因cDNA片段，连接到pEASY-Blunt载体上，转化到Trans1-T1感受态细胞内，筛选阳性单克隆进行菌落PCR鉴定并测序验证；利用限制性内切酶双酶切重组载体获得含有黏性末端的目的基因片段，并对原核表达载体pET-32a＋和pGEX-4T-1进行完全双酶切，连接目的基因片段后获得重组原核表达载体，将重组原核表达载体转化至大肠杆菌原核表达菌株BL21（DE3）；IPTG诱导表达获得目的蛋白，并对蛋白表达条件（诱导时间、诱导温度及IPTG浓度等）进行优化，鉴定表达蛋白水溶性。
문장제취의남개식주총RNA ，통과RT-PCR확증출AtMYB50여AtMYB61기인cDNA편단，련접도pEASY-Blunt재체상，전화도Trans1-T1감수태세포내，사선양성단극륭진행균락PCR감정병측서험증；이용한제성내절매쌍매절중조재체획득함유점성말단적목적기인편단，병대원핵표체재체pET-32a＋화pGEX-4T-1진행완전쌍매절，련접목적기인편단후획득중조원핵표체재체，장중조원핵표체재체전화지대장간균원핵표체균주BL21（DE3）；IPTG유도표체획득목적단백，병대단백표체조건（유도시간、유도온도급IPTG농도등）진행우화，감정표체단백수용성。
Total RNA was extracted from A rabidopsis seedlings ,and cDNA fragments of A tMYB50 and A tMYB61 genes were amplified by RT-PCR .cDNA fragments were subsequently cloned into pEASY-Blunt vectors ,and then were transformed into Trans-T1 phage resistant chemically competent cells .The analysis of bacterial colony PCR and cDNA sequencing were performed to confirm that cD-NA of the A rabidopsis thaliana A tMYB50 and A tMYB61 genes was successfully cloned .Their cDNA fragments with sticky ends were isolated by using restriction enzyme digested .The cDNA fragments were cloned into prokaryotic expression vectors pET-32a+ and pGEX-4T-1 .In addition ,the recombi-nant plasmids were transformed into BL21(DE3)plysS chemically competent cells .The expression of MYB61 and MYB50 was induced to generate their fusion proteins by IPTG .The protein expression conditions such as the inducing time and temperature and the IPTG concentration were optimized ,and the water solubility of the protein was tested .