Effects Of The Expression Of Cation/H~+ Antiporter Gene TsCHX18 From Thellungiella Salsugineain On The Salt Tolerance Of Maize

Salinity is a major abiotic stress and saline-alkali soil accounts for approximately 7%of the world's total land area.The reduction of crop production caused by salt stress losses billions of dollar every year.The maize(Zea mays L.)is a high yield crop,and it is not only an important crop but also a crucial material of industry.Plants have two discrete mechanisms response to salt stress.One of them is the biosynthesis of compatible solutes,such as the proline and mannitol.These solutes can keep a stable cellular osmotic pressure to resist osmotic stress when plants surf erring from salinity environment.However,de novo synthesis of compatible solutes is a relatively slow process to relieve the salt stress and it has little effects on the ionic toxicity.Another mechanism is maintaining ionic homeostasis in cell under salty conditions.Many kinds of proteins regulate the ionic balance in the plants,such as H+-ATPase,H+-PPase,secondary transporters and ion channel proteins.Potassium ion(K+)is one of the most abundant cations in plants.K+ absorption and transportation play very important roles in maintaining normal K+/Na+ ratio in cells.There are a large number of genes encoding potassium ion transporters and potassium channel proteins,which uptake K+ from the environment and transport them among different tissues in plants.There are two kinds of K+ uptake mechanisms,one is called the low affinity potassium transport mechanism(mainly performed by K+ channel proteins),another is called the high affinity potassium transport mechanism(mainly performed by K+ transporters).For the later,there are four encoding gene families:TrK/HKT(high-affinity potassium transporter),KEA(K+/H+ antipoters)and KT/KUP/HAK(K+transporter/K+ uptake permease/high-affinity K+ transporter).CHX(cation/H+ antiporters)is a kind of cation transporters playing important roles in responding to adversity environment in eucaryon.In this study we transformed the gene TsCHX18 from halophyte Thellungiella salsuginea into the maize inbred lines DH4866 with the Agrobacterium strain LBA4404 harboring the Mini-Ti plasmid pCAMBIA3300-P35S-bar-P35S-TsCHX18 to infect the shoot tip of the seedlings,and the transgenic homozygous lines were selected by the herbicide Basta(glufosinate)resistance and PCR detection.The positive plants in each generation were transplanted in the field to produce the seeds by self-pollination.The results of semi quantitative RT-PCR and qRT-PCR detection of the T2 generation homozygous lines transgenic showed that TsCHX18 was active and stable expressed in the transgenes maize plants in successive generation,which indicated that the gene had integrated into the maize genome.Among them,three lines from different independent transgenic events were selected for resistance evaluation and yield determination,with the transgenic receptor inbred lines as controls.Three transgenic lines were selected for the evaluation of salt tolerance at seed germination and seedling stage,with receptor inbred lines as control.When the seeds of transgenic and control lines were germinated in the absence of NaCl,germination rate and germination potential were not different among the lines.In the culture with 100 mM or 150 mM NaCl nutrient solution,the germination rate and germination potential of transgenic lines were significantly better than the controls.The root development of the transgenic lines was much better than that of the controls.The hydroponic culture plants at two leaf stage,which is the most sensitive stage of maize plants to salt stress,were treated with 100 mM NaCl for 2 weeks.The transgenic lines were only slightly damaged,and the root length and lateral roots of which were well-developed,while the leaves of the control lines showed wilting symptom,and the growth of roots was heavily inhibited,namely TsCHX18 transgenic maize seedlings improved the salt tolerance in salinity environment.The biomasses and the growth performance of transgenic plants were much higher and much better than those of the control line under salt stress conditions.After suffering from salt stress,the MDA content and electrolyte leakage of the transgenic lines were significantly lower than the control line,which suggested the cells of transgenic lines had less damage of membrane system.At the same time,the leaves of transgenic lines accumulated more osmolyte solutes,and the contents of proline and sugar were significantly higher than those of the controls,suggesting the ability to maintain the osmotic potential and water absorption were better.Under NaCl stress conditions,the transgenic maize showed higher K+ absorption capacity,which significantly increased the concentration of K+ in cells,thereby the stability of the K+/Na+ ratio was maintained to enhance the continued growth of plants.In the field,the plants of transgenic lines showed similar phenotypes and growth rate and disease resistance compared with the control line.In coastal saline soil the transgenic lines significantly improved salt tolerance and ear traits,and the ear length,kernel number and 100 grain weight of each line were significantly better than those of the control line.In summary,the active expression of TsCHX18 in maize can significantly improve the salt tolerance of plants,and these transgenic maize lines have good practical applications in the future.