Characteristics Of Over-Expression For Nitrate Transporter Genes OsNRT1.2 And OsNRT1.5 In Rice

Nitrogen (N) is considered as an important limited factor of crop yields. Both ammonium and nitrate are the major sources of soil N for plants. Rice (Oryza sativa L.) is not only the major food for a large part of world population, but also an important model plant for monocot species research. More and more studies have reported that nitrate transporters have been proved very important for rice growth. Although the genome size of rice is small and its genome sequence was completed, it has few reports about the physiological and molecular mechanism about how rice takes up nitrate.It has been found some nitrate transporters belonging to the low affinity nitrate transporter (NRT1) family in Arabidopsis. Two transporters CHL1 and AtNRT1.2 have been shown the function in nitrate uptake, AtNRTl.4 involved in nitrate storage in petioles, AtNRT1.7 playing a role in nitrate remobilization from older to younger leaves,and AtNRT1.6 involved in delivering nitrate for seed development. In addition, AtNRT1.5 mediated nitrate efflux in cortex nitrate export and xylem loading.In this study, two putative NRTl genes, OsNRT1.2 and OsNRT1.5, the homologous of AtNRTl.2 and AtNRTl.5 in rice, were found basing on the rice genomic database and bioinformatics analysis. By agrobacterium tumefaciens transformation, the gain of function of rice OsNRT1.2 and OsNRT1.5 genes was tested by over-expression in Wuyunjing 7 (Wyj7) and Nipponbare rice cultivars. Wild type of Wyj7 (WWT) and Nipponbare(WT) as control, the trasgetic lines for futher study, such as W1.2-OE1,W1.2-OE2,W1.2-OE8 and W1.5-OE2,W1.5-OE7,W1.5-OE8 from Wyj7,1.2-OE1,1.2-OE5,1.2-OE6 and 1.5-OE1, 1.5-OE9,1.5-OE16 from Nipponbare.The main results were as follows:1) The OsNRT1.2 cDNA encoded 593 amino acids with full-length of 2178 bp and open reading fragment (ORF) of 1781 bp, while OsNRT1.5 cDNA encoded 610 amino acids with full-length of 2240 bp and ORF of 1833 bp. The classification of OsNRTl.2 and OsNRT1.5 amino acids sequence was a nitrate transporter gene and their putative structure was predicted to be a membrane protein with 12 cross membrane motifs and a center hydrophilic loop. OsNRTl.2 was composed by four exons and three introns, the same as OsNRT1.5.2) The expression pattern of OsNRTl.2 in Wyj7 and Nipponbare was investigated and the results showed that its mRNA was only expressed in roots but not in shoots. Moreover, the expression of OsNRT1.5 in Nipponbare was in shoots more than in roots.3) Comparison with Wild type, the expression of OsNRTl.2 and OsNRTl.5 in transgenic lines increased, while there was no difference between different nitrate concentration.The expression of OsNRT1.1, OsNRT2.1 and OsNRT2.2 did not change in the shoots and roots of transgenic lines.4) Compared with WWT, W1.2-OE1, W1.2-OE2 and W1.2-OE8 were used in growth experiment at 5.0 mM nitrate conditions for 30 days, the plant height of them increased by from 3.1 cm to 3.7 cm, while the root length, nitrogen content and N uptake of transgenic lines significantly was increased by 3.3 cm,2.5 cm,4.5 cm; 11.3%,10.9%,14.6% and 39.5%,33.9%,97.8%. While plant height, Nitrogen (N) content in shoot and root was no difference between WWT and transgenic lines at 0.2 mM NO3-. But at 5.0 mM NO3-, compared with WWT, the shoot nitrogen content of W1.2-OE2 and W1.2-OE8 except W1.2-OE1 was increased.The dry weight of transgenic lines was significantly increased by from 9.4% to 31.1% at 0.2 mM nitrate and from 12.5% to 43.8%.The dry weight in shoots and roots of W1.2-OE1, W1.2-OE2 and W1.2-OE8 significantly increased by 35.4 %～57.2% and 79.3%～120.9% in field. The present study reported that OsNRTl.2 in Wyj 7 may be involved the N uptake from plant roots and therefore more shoot biomass was increased.5) There was no difference for root length between transgenic lines of OsNRTl.2 over-expressed in Nipponbare at 0.2 mM NH4+,5.0 mM NH4+,0.2 mM NO3-,5.0 mM NO3-and 2.5 mM NH4NO3.However, the shoot height of WT,1.2-OE1,1.2-OE5,1.2-OE6 increased by 7.8 cm,20.2 cm,18.6 cm,27.2 cm at 2.5 mM NH4NO3. Compared with WT, the shoot nitrogen content of 1.2-OE1,1.2-OE5,1.2-OE6 increased by 27%,18%,23% at 5.0 mM NH4+and 15.6%,9.5%,19.2% at 2.5 mM NH4NO3. The number of effective tiller for transgetic lines was increased in field, while there was no difference for dry weight of shoots and roots. 6) Comparison with WWT, the dry weight of W1.5-OE2, W1.5-OE7, W1.5-OE8 increased by 54.8%,74.6%,36.7% at 5.0 mM NH4+ and 68.5%,24.8%,31.5% at 2.5 mM NH4NO3.Transgenic lines of OsNRT1.5 over-expressed in Wyj 7 early tillered and the roots became stronger and better at 5.0 mM NO3-,2.5 mM NH4NO3 and 5.0 mM NH4+.Moreover, transgenic lines of OsNRT1.5 early tillered in field. Grain weight per plant expect W1.5-OE7, setting rate and setting number per tiller increased significantly, while there was no difference for plant height, tiller number expect W1.5-OE2, effective spike number and grain weight. The present study reported that OsNRTl.5 in Wyj 7 may be involved the N translocation from plant roots to shoots, improved tiller and therefore more grain weight per plant was increased.7) Compared with WT, the shoot nitrogen content of 1.5-OE9 increased by 13.3% at 5.0 mMNH4+ and the shoot nitrogen content of 1.5-OE1,1.5-OE9,1.5-OE16 increased by from 14.5% to 38.2% at 2.5 mM NH4NO3. While there was no difference for the shoot nitrogen content between transgenic lines and WT at 0.2 mM NO3-,5.0 mM NO3-,0.2 mM NO3-.Morever, the plant height of Nipponbare transgenic lines decreased, except 1.5-OE16, while setting rate and setting number per tiller increased significantly, and there was no difference for grain weight per plant, tiller number expect 1.5-OE1, effective spike number and grain weight and shoot dry weight exceptl.5-OE9 in field.