Cloning And Sequence Analysis Of Potassium Channel Gene NKT5 In Nicotiana Tabacum And Construction Of Transgenic Vectors

Potassium has an important influence for yield and quality of tobacco, and potassium can increase resilience of tobacco. Potassium ion channel is the major channel to absorb potassium for tobacco. In this research, potassium channel gene NKT5 was cloned from ordinary tobacco by RACE, and sequence analysis and bioinformatics analysis were carried on; and then the transgenic vectors of NKT5 were built, to provide technical support and theoretical basis for further research.In this research, a pair of specific primers was designed based on the 490 bp middle segment of K+ channel gene to acquire 5'and 3'-ends of cDNA by RACE strategy. An unreported K+ channel gene in tobacco named NKT5(NCBI accession number: HM010922)was obtained by splicing three cDNA sequences and the identification of full-length cloning.The full-length cDNA of NKT5 is 2365bp long with 190bp 5'-untranslated region, 249bp 3'-untranslated region and 1926bp coding sequence. The deduced amino acid is 641 AA long. Through the analysis of the conservative domain, transmembrane domain and spatial structure of protein, the related bioinformatics analysis of the potassium channel gene NKT5 were obtained; and a phylogenetic tree of Kï¹¢channels from Nicotiana tabacum, Arabidopsis and related plants was constructed. In this paper, the gene sequence analysis and function prediction were carried out, in order to clarify molecular mechanisms of Potassium absorption and stress-resistance in the tobacco. The research provides the basis for the study of functional expression.To nucleotide sequence of the potassium channel gene NKT5 as a template,By specific primers designed with Restriction sites and RT-PCR method, full-length NKT5 was cloned from ordinary tobacco. Through sequencing analysis, its sequence is consistent with NKT5 reported. Connected NKT5 with pCAMBIA1300, pCAMBIA1302 treated with digestion, it is proved to acquire successfully the transgenic expression vectors after connecting, transformation, PCR and restriction digestion analysis. Then transformed vectors constructed into Agrobacterium, they can be used in subsequent transgenic experiments to reveal the potassium absorption characteristics of tobacco and resistance mechanisms.