琯溪蜜柚汁胞Beta-tubulin cDNA的克隆及序列分析

以琯溪蜜柚汁胞为材料,采用cDNA克隆方法进行Beta-tubulin cDNA的克隆和表达.结果表明:琯溪蜜柚果肉Beta-tubulin cDNA全长1636 bp,有完整的阅读框,编码444个氨基酸;5′非翻译区60 bp,3′非翻译区243 bp(不包含终止密码子TAA),其中包括1个多聚腺苷酸化信号AATAAA以及1个含21个腺苷酸的poly(A)尾.由琯溪蜜柚果肉Beta-tubulin cDNA推导的氨基酸序列与陆地棉(Gossypium hirsutum)、大豆(Glycine max)、杨属植物(Populus)等的同源性较高,分别为86%、85%、84%等.通过构建重组质粒p28-PTU,并转化至Trans Rosetta(DE3)中,在诱导温度为37℃、IPTG浓度为1 mm、诱导时间为4 h的条件下,其在大肠杆菌宿主中获得了表达.

Cloning and sequence analysis of Beta-tubulin cDNA of juice sac in Citrus grandis (L.) Osbeck

The pummelo(Citrus maxima(L.) Osbeck) juice sac Beta-tubulin cDNA was cloned,sequenced and analyzed.The results showed that the complete cDNA was 1636 bp in length,including 60 bp 5′ untranslated region,243 bp 3′ untranslated region which contained a polyadenylation signal AATAAA and a 21 bp polyadenylation tail.The coding region encoded a peptide of 444 amino acid residues.The deduced amino acid sequence deduced by the Beta-tubulin sequences of juice sac in Citrus grandis(L.) had higher homology with upland cotton(Gossypium hirsutum),fig(Ficus carica) and poplar(Populus),which were 86%,85% and 84%,respectively.The coding region of the cDNA was ligated into pET-28a vector to build a recombinant vector p28-PTU,which was expressed in Escherichia Coli Trans Rosetta(DE3) strain when induced with 1 mm IPTG and grown for 4 hours at 37 ℃.