Cloning, Expression Of A Novel Nitrate Transporter Gene OsTNrt2.1 In Rice And Gene Genetic Transformation

Nitrate (NO₃^-)is one of the major sources of nitrogen for crops. Deficiency of nitrateleads to growth inhibition of plants. Meanwhile, leaching of nitrate in soils could results inthe eutrophication nutrition of underwater and be harmful of the people's health. Therefore,improvement of the utilization efficiency of nitrate fertilizer will contribute to the highefficiency of natural resource and reduce the environment contamination. Based on theOsNrt2.1 cDNA sequence of rice (Oryza sativa) variety Nipponbare, a nitrate transportergene OsTNrt2.1 with high similarity with OsNrt2.1 was cloned from TP309, a rice varietywith high nitrogen use efficiency. The structure, characterization and the expression patternof OsTNrt2.1 were elucidated in this study. Meanwhile, the binary vector ofpCAMBIA1305-OsTNrt2.1and pCAMBIA1305-OsTNrt2.1, fused the ORF of OsTNrt2.1and the promoter region of OsTNrt2.1, respectively, were constructed. The constructedbinary vectors were used to transform the tobacco plants based on the transformationmethod mediated by Agrobacterium tumefeciens (Strain LBA4404). The transgenic plantsfused OsNrt2.1 and OsNrt2.1 promoter were generated, which provide a platform foridentification of OsNrt2.1 in nitrate transport capacity and exploration of OsNrt2.1regulation mechanism in expression in future.The mainly results in this study are as follws:1. By searching the whole genome datebase of rice in website NCBI, a rice putativenitrate transporter gene OsNrt2.1(GenBank accession No: AB008519) was identified. Thecorresponding DNA sequence is searched by BLAST analysis of OsNrt2.1. The forwardand reverse primer were designed based on the 5' and 3' flanking region sequence ofOsNrt2.1, which were used to PCR amplify this novel nitrate transporter gene with thetotal DNA of TP309 being template One about 1.7 Kb cDNA fragment was amplified andcloned in pUCm-T vector. The plasmid from positive DH5αclone was sequenced and theinsertion was 1707 bp. 2. It was found that there were two bases difference between the novel gene andOsNrt2.1 with a identity of 99.6%. Therefore, it was named OsTNrt2.1. The full length ofOsTNrt2.1 cDNA is 1928 bp, including the ORF 1602 bp and 5' untranslated region of 96bp and 3' untranslated region of 230 bp, respectively.3. The translated protein of OsTNrt2.1 was characterized by protein analysis softwareExPASy. OsTNrt2.1 encodes 533 amino acids. The molecular weight and isoelectric pointof OsTNrt2.1 are 57.2 KD and 8.12. A transmembrane predication analysis explored thatOsTNrt2.1 has 12 transmembrane domains, with a center loop consiting of 24 amino acidsand a long C-terminal. A signature motif (A-G-W/L-G-N-M-G) of NNP was found intransmembrane domain fifth. OsTNrt2.1 also had six conserved protein kinase C motifs(S/T-X-R/K), being separately distributed in non-transmembrane domains.4. Under the conditions of different nitrogen sources and concentrations in themedium, there were always much more transcripts in roots, suggesting that OsTNrt2.1 hasimportant functions in NO₃^- uptake for roots at different nitrogen levels in the growthmedium.5. The expression of OsTNrt2.1 was inhibited by NH₄⁺ and induced by NO₃^- in themedium. The expression pattern of OsTNrt2.1 was affected by photoperiod, showing atypical diurnal regulation with high expression level in the light period. The transcripts ofOsTNrt2.1 were similar under different NO₃^- concentrations, suggesting that OsTNrt2.1 ispossibly a dual-affinity nitrate transporter.6. Based on routine molecular cloning techniques, the plant binary expression vectorpCAMBIA1305-OsTNrt2.1 fused the ORF of OsTNrt2.1 with correct frame inpCAMBIA1305 was constructed. The binary vector pCAMBIA1305-OsTNrt2.1 was usedto transform Agrobacterium Tumeficiens LBA4404. A positive LBA4404 clone was used totransform tobacco by leaf-circle method. Some transgenic plants were generated for furtherevaluation of nitrate transporatation.7. The promoter region locating at the 5' flanking region of OsTNrt2.1 was PCRamplified based on the sequence of AP004752 using TP309 DNA as template. Thefragments length range from 173 bp to 2047 bp. Based on molecular cloning techniques,the plant binary expression vector pCAMBIA1305-OsTNrt2.1 which fused OsTNrt2.1promoter was constructed. The positive LBA4404 colony fused OsTNrt2.1 promoter wastransformed tobacco by leaf-circle method. The transgenic tobacco plants were screenedand generated for further study of the regulatory elements which regulate the OsTNrt2.1 expression. The purpose of this study is aimed at providing a basis for fine identifyingregulatory elements which control the gene expression under nitrogen stress.