烟草转酮醇酶基因(NtTK)的克隆与表达

Cloning and Expression of Transketolase Gene(NtTK)in Tobacco

根据烟草（Nicotiana tabacum）转酮醇酶（transketolase）基因序列设计引物，通过RT-PCR克隆了珊西烟（Nicotiana tabacum L．cv．Xanthi NN）的transketolase基因，并将其命名为NtTK．该基因全长2235bp，编码744个氨基酸，与已报道的烟草TK基因（A52295）的核酸序列同源性迭99％，与TK基因氨基酸序列一致性达97．16％，有18个不同氨基酸残基，尤其在第101—110位点有连续10个氨基酸残基完全不同．利用pMAL—c2x原核表达载体对transketolase基因进行了原核表达，并获得纯化的蛋白．

Primers were designed according to the sequence of the transketolase gene （A 52295） of Nicotiana tabacum, and a Nicotiana tabacum L. cv. Xanthi NN transketolase gene named NtTK was cloned by RT-PCR. NtTK is 2 235 bp long and encodes 744 amino acids. It is 99% identical to the reported TK gene of Nicotiana tabacum, and the amino acids of NtTK is 97. 16% identical to A 52295. There are 18 amino acids that are different from A 52295, especially residues 101 -110 are completely different. NtTK was cloned into pMAL-c 2 x vector and transformed into the E. coli TB 1 strain in order to express and purify NtTK protein.