Cloning Of NHXs From Halophytes And Construction Of RNAi Vectors For KcNHXs And Their Transformation Of Karelinia Caspica

Na⁺/H⁺ antiporters on the tonoplast have been suggested to play a major role in the maintenance of osmotic homeostasis when plants are suffered from salt stress. Na⁺/H⁺ antiporters transport Na+ into the vacuole with the electrochemical gradient of protons generated by the vacuolar H⁺-translocating enzymes, H⁺–adenosine triphosphatase (ATPase) and H⁺–inorganic pyrophosphatase (PPase). The mechanism of compartmentation of Na⁺ into vacuoles through the operation of a vacuolar Na⁺/H⁺ antiporter provides an efficient way to avert the deleterious effects of Na+ in the cytosol and maintains osmotic balance to drive water into the cells.Populus euphratica Oliv is famous for growing in the extremely saline, arid environment and it's reported that there is a coordinated relation between Na+-transporters on vacuole membrane and plasma membrane when plants are exposed to different concentration of NaCl solution. So the regulation mechanism of genes encoded Na+-transporter on vacuole membrane and plasma membrane deserves studying.Karelinia caspica seedlings can become succulent under salt stress, while it is a typical recretohalophyte. The roles of NHX in the succulence and salt-secreting of seedlings are worthy to study.According to the conserved region of homology sequences, specific primers were designed, and combining with the technology of RT-PCR and RACE, two members of Na⁺/H⁺ antiporter family of Karelinia caspica (KcNHX1 and KcNHX2) and one member of Na⁺/H⁺ antiporter family of Populus euphratica Oliv (PeNHX2) were cloned, whose GenBank accession numbers are DQ303231, DQ304690, DQ414512 respectively.To testify whether KcNHXs are salt-associated genes, we did the semi- quantitative RT-PCR of KcNHX1 and KcNHX2, and the results showed that the expression of KcNHX1 and KcNHX2 mRNA in plants was both induced when they are treated with 300 mmol·L^-1NaCl, while their expression was suppressed under 500 mmol·L^-1 NaCl. The study also found that basal expression of KcNHX1 was more than that of KcNHX2, which indicated that KcNHX1 and KcNHX2 might carry on different physiological roles respectively in the course of growth and development of Karelinia caspica.To further elucidate the roles of KcNHXs in Karelania caspica under salt stress, on the base of establishment of tissue culture of Karelania caspica(media of inducing shoots:MS+6-BA 0.4 mg·L^-1+NAA 0.5 mg·L^-1;media of inducing roots:1/2MS+NAA 0.2 mg·L^-1)and the two cloned KcNHX genes, we designed intron- mediated RNAi vector with sense and antisense strand of KcNHX1 and KcNHX2 respectively, which could form hair-spin constructs that had been reported to interfere gene's function more efficiently compared with antisense constructs and co-suppression constructs. Then Karelinia caspica was transformed with the RNAi vector by the method of leaf-disc cocultivation. Genome of hygromycin-resistant plants were extracted and testified by PCR, the results showed that the RNAi elements of KcNHX1 and KcNHX2 had been integrated into the genome of Karelinia caspica. The study provides foundation for further study of the roles of NHXs in Karelinia caspica throungh RNAi techenology.