Functional Analysis Of The Vacuolar Na~+/H~+ Antiportor (NHX) Gene In Kareliana Caspica By Using RNA Interference Technology

Too much of Na⁺ ion is harmful to plants,while plants,with the energy provided by transporting proton with its concentration gradient,vacuolar-type Na⁺/H⁺ antiporters delivered Na⁺ into the vacuoles against the electrochemical gradient generated by vacuolar H⁺-ATPase and H⁺-PPase.This compartmentation of Na⁺ provided an effective mechanism to avert the deleterious effects of Na⁺ in cytosol and maintain an osmotic potential that increases the ability of holding the cell moisture and enhances the salt tolerance.RNA interference means short double strand RNA induces the homologous RNA breaking up and leads to the phenomena of expression silence and fuction losing of the target gene.RNA interference(RNAi) is now widely used in plant biotechnology,both as a useful tool for discovering or validating gene functions as well as a quick way of engineering specific reductions in expression of chosen genes.RNA interference(RNAi) technology has already been applied to silence gene expression in a variety of organisms.Karelinia caspica is a perennial herbaceous plant species.It commonly distributes on salt meadow or salina.K.caspica belongs to salt-excreting species.In the previous work,our laboratory has successfully established K.caspica tissue culture system,and then cloned cDNA sequence of vacuolar-type Na⁺/H⁺ antiporters gene KcNHX1,KcNHX2(DQ303231, DQ304690) from K.caspica in Xinjiang.Result showed that the homology was not much high between KcNHX1 and KcNHX2 genes(64.91%identities),both gene belong to the different members of NHX genes family,and they may play different physiological functions. In order to investigate the idiographic different physiological functions of KcNHX1 and KcNHX2 contributed in salt tolerance process of K.caspica.RNAi technology was used in construction RNAi vectors,and partial coding sequence of KcNHX1 or KcNHX2 and a 120 bp intorn sequence which induces to form partnership structure from KcNHX2 were choosed as RNAi target sites. Based on above results,both the pCAMBIA 1301-1.1-KcNHX1i and pCAMBIA1301-1.1 -KcNHX2i were introduced into K.caspica by Agrobacterium-mediated method,T₀ homogenous plantlets were obtained from hygromycin-resistant seedlings.PCR analysis indicated that KcNHX1i and KcNHX2i were integrated into the genome of K.caspica.To transgenic plants were confirmed by analyses of RT-PCR and salinity resistance.The results indicated that 4 out of 6 of the To transgenic plants of pCAMBIA1301-1.1-KcNHX1i showed NHX1 gene being silenced,and the 4 out of 8 of the To transgenic plants of pCAMBIA1301-1.1 -KcNHX2i showed NHX2 gene being silenced.Salinity resistance test of transgenic plants indicated that RNAi technology is efficient for gene silencing in plants.The MDA and relative electric conductivity of the transgenic plants were higher compared to WT under NaCl treatment,the chlorophyl of the transgenic plants of pCAMBIA1301-1.1-KcNHX1i was lower.The antioxidation of the transgenic plants of pCAMBIA1301-1.1-KcNHX1i was weaker than the WT.This indicated KcNHX1 gene might be very important in salt tolerance of K.caspica.But the results of MDA and the chlorophyl of transgenic plants of pCAMBIA1301-1.1-KcNHX2i did not show in the logic way,The salt tolerance of plants are not changed.so the function of KcNHX2 gene in salt tolerance needs further study.