| Potassium is the second important mineral nutrient for plants,and essential for plant growth and crop production.Soil salinity is one of the major abiotic stress restricting agricultural productivity and ecological environment worldwide.Due to the similarity between Na+ and K~+ in terms of structure and chemical properties,excessive Na+ in plant could disturb K~+ uptake and inhibit plant growth,even lead to plant death.During long time evolution,halophytes formed specialized adaptation mechanisms to respond salt stress.Systematical understanding the salt-tolerant mechanism of these plants,screening and cultivating salt-tolerant plants is important for improving saline-alkali land ecosystem as well as taking advantage of land resources.As a typical salt-excluding halophyte,the salt-tolerant mechanism of Puccinellia tenuiflora is limiting Na+ unidirectional influx in roots and reducing the accumulation of Na+ in plant to maintain a strong selectivity of K~+/Na+.Thus,P.tenuiflora has a strong ability to regulate K~+,Na+ homeostasis.However,there are few systematical studies about the uptake and transport of K~+ in P.tenuiflora.In present work,we established the genetic transformation system of P.tenuiflora,cloned PtHAK5 of KT/HAK/KUP family through RT-PCR technology and verified the function characterization of the protein by yeast heterologous expression method.We obtained following conclusions:1.The optimization of a high frequency regeneration system of P.tenuiflora(1)5.0 mg·L-1 2,4-D and 0.5 mg·L-1 KT are the optimal hormone combination for callus induction,reducing the concentration of 2,4-D to 2.5 mg·L-1 is beneficial to improve the differentiation rate of callus during a period of subculture culture;0.5 mg·L-1 NAA,2.0 mg·L-1 6-BA and 2.0 mg·L-1 KT are the best hormone combination for callus differentiation,which could significantly increase the rate of callus regeneration.(2)The appropriate amount of organic compounds(100 mg·L-1 Hydrolyzed casein,500 mg·L-1 Proline and 300 mg·L-1 Glutamine)and 0.3% phytagel could inhibit vitrification of callus and significantly increase the rate of embryogenic callus.2.The establishment of a high efficiency genetic transformation system of P.tenuiflora(1)When callus was sub cultured 3 to 4 times and then pre-cultured on MS medium supplemented with 10 g·L-1 glucose and 200 ?M·L-1 AS for 4 days to infect,the structure of callus was favorable for integrating exogenous DNA,the abilities of callus to resist injury caused by Agrobacterium tumefaciens and recover from transformation were strong.(2)Per-cultured callus was infected by A.tumefaciens strain GV3101 with OD600 = 0.6 for 15 min,then co-cultivated with GV3101 on MS medium supplemented with 200 ?M·L-1 AS for 2 days.It is the optimal infection condition of genetic transformation in P.tenuiflora.(3)Co-cultured callus was washed by 250 mg·L-1 Cef solution for 20 min,and then inoculated on MS medium supplemented with 250 mg·L-1 Cef,which has a good effect on antibacterial.After bacteria-elimination,the resistant callus was screened on differentiation medium containing 50 mg·L-1 Kan.3.The gene PtHAK5 encoded by high-affinity K~+ transporter was cloned from P.tenuiflora,the length of open reading frame(ORF)was 2355 bp,the ORF sequence encodes 784 amino acids contained 11 transmembrane regions,the homology between PtHAK5 and other high-affinity K~+ transporter from high plant was higher,it is the member of cluster?in KT/HAK/KUP family.4.Overexpressing PtHAK5 in yeast mutant,the growth of yeast mutant CY162 defected K~+ uptake function was recovered under low K~+ conditions and without increasing the salt sensitivity of mutant strain G19 deficient Na+ efflux,indicating that PtHAK5 only mediates K~+ uptake,was not participated in Na+ transport. |
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