The Cloning And Expression Regulation Of Potassium Uptake Relative Genes In Nicotiana Tobacum

Except for affecting the disease resistance and stress responses in tobacco growing periods, the potassium nutrition has close relations with quantity and quality of flavor, burn capacity and the amount of harmful materials such as tar and benzopyrene. The potassium content in tobacco leavies is an important index for high quality tobacco leaf. Surveys have confirmed that potassium content of domestic tobacco leaf is much less than that of international high quality tobacco leavies from USA, Zimbabwe and Brazil. Researchers from home and abroad have done many studies to increase the potassium contents in tobacco leaf through raising fertilizer amounts or improving fertilization methods in recent years. But unfortunately, all the works have failed to increase the potassium of tobacco leavies. Low potassium content in tobacco leaf is an important technique problem which puzzled growers in domestic tobacco growing areas.Three genes including AtKup1 encoding the K~+ transporter, AtNHX1 encoding the Na~+/H~+ anti-porter and inorganic pyrophosphatase AVP2 have been cloned from Arabidopsis. The plant binary expression vectors of the 3 genes have been constructed and transformed into tobacco by the meditation of Agrobacterium tumefaciens. The transcript of 5 tobacco internal genes encoding K~+ channel, K~+ transporter, vacuole H~+-PPase, plasma H~+-ATPase and vacuole H~+-ATPase have been analyzed in transgenic tobacco plants. The results demonstrated that the transcript of NtHAK1 gene was reduced and that of NHA1 was increased significantly in the roots of the AtKup1 trans-formants. The transcript of NtHAK1 gene was also down regulated in the roots of AtNHX1 trans-formants, but the transcripts of NHA1 and VAG1 encoding H~+-ATPase were up regulated significantly and that of NVP1 encoding vacuole H~+-PPase was increased slightly. The transcript of VAG1 encoding H~+-ATPase and NVP1 encoding for vacuole H~+-PPase are down regulated and the transcript of NtHAK1 are up regulated significantly and that of K~+ channel NKT1 are increased slightly in the tobacco root over expressed of AVP2 gene. The chemical contents analysis of tobacco leaf indicated that the potassium uptake capacity of trans-formant roots were increased differently when the tobacco was genetically modified by the three genes above respectively and the potassium contents in the tobacco leaf were increased differently.PCR-based DNA shuffling was carried out for AtKup1 gene to recombinatorial molecular evolution in vivo. The AtKup1 mutations with high capacity of K~+ uptake were screened from recombination PCR fragments libraries and introduced into TRK1 and TRK2 defective yeast strain.By the bioinformatics analysis of genes encoding to K~+ high affinity transporter KT/HAK/KUP, potassium channel, H~+-ATPase and vacuole H~+-PPase from Arabidopsis and other species in NCBI. Four cDNA complete sequences incuding NrHAK1, NKC1, NVP1 and NtHAK1 were obtained through the methods of degenerate primers PCR and RACE, The nucleotide sequence have been deposited in the NCBI data base. Function characterization of NrHAK1 which was carried out by introduced it into the K~+ uptake genes TRK1 and TRK2 defective yeast strain demonstrated that NrHAK1 can complement yeast mutants defective in K~+ uptake. It was found that the mRNA of NrHAK1 was the most abundant in roots of Nicotiana rustica which suggest that NrHAK1 plays an important role in potassium uptaking of plant roots.The qRT-PCR technique which suit for mRNA measurements in the molecular nutrition studies was established by performing the optimized experiments of primer designation, PCR systems and condition as well as house keeping gene selection. Transcript of five genes related to the potassium uptake including K~+ channel, K~+ transporter, cytoplasm H~+-ATPase, H~+-PPase and vacuole H~+-ATPase under different nutrition and salts stress conditions were analyzed by using qRT-PCR methods. It was found that the transcript of NtHAK1 and NHA1 were significantly stimulated and that of NVP1 was reduced when the K~+ starvation exist in external solution. The results also confirmed that the transcript of K~+ channel gene NKT1, K~+ transporter gene NtHAK1, V-H~+-ATPase G unit gene VAG1 and P-H~+-ATPase gene NHA1 were down regulated when the tobacco was Ca2~+ starved. The transcript of K~+ transporter gene NtHAK1 was induced by excessive Na~+ salinity stress, but that was inhibited when tobacco plant exposed to the solution contained 5 mmol/L NH4~+.