Cloning Of Atnhx1 And Hvbadh1 Genes And Transformation Of 3 Plant Species

Salinity is a major constraint to plant productivity. In order to adapt to the salt stress, there are two common mechanisms found in plants, one of which is to rebuild the cellular ion homeostasis, the other is to accumulate the compatible solutes. Rebuilding the cellular ion homeostasis involves the intracellular regulation of absorbing Na⁺, K⁺ and Cl^- as well as subsequent vacuolar compartmentalization to prevent the accumulation of toxic ions in the cytosol. Vacuolar sequestration of Na⁺is an important and cost-effective strategy for osmotic adjustment that also reduces the Na⁺ concentration in the cytosol. Na⁺ sequestration into vacuolar depends on the expression and activity of Na⁺/H⁺ antiportors. On the other hand, the compatible solutes, including betaines and related compounds, polyols and sugairs (such as mannitol, sorbitol, and trehalose), and amino acids (such as praline), are accumulated differently among plant species. Among them, betaines appear to be a critical determinant of stress tolerances.In this study, the open reading frame(ORF) of atnhx1-cDNA, encoding the A. thaliana vacuolar Na⁺/H⁺ antiportor, was cloned by using one-step RT-PCR from the Arabidopsis thaliana seedlings after 24h treatment with 100 mmol/L NaCl. Two primers for PCR were designed following the atnhx1 sequence from NCBI (accession number: AF 510074). The atnhx1-cDNA we obtained is 1,617 bp, which just was an open reading frame encoding a predicted polypeptide of 538 amino acids, and was high homologue (99.7%) to the original atnhx1 sequence. In order to construct an expression vector which can express atnhx1-cDNA in plant, an expression cassette, including a CaMV35S promoter, aΩfragment of TMV RNA 5'UTR, atnhx1-cDNA and a NOS polyA sequence, was introduced into the T-DNA region of the binary vector pNT. The plant selectable marker gene of this recombined vector is nptII delivering kanamycin resistance to the transgenic plants. This recombined binary vector named as pNT-atnhx1 was transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, which could be used for plant transformation directly.In the experiment, a highly-efficient and stable protocol for Agrobacterium mediated transformation was established and was adapted for the genetic transformation of two plant species, Astragalus melilotoides Pall and Cichoriuminty bus L. Five factors affecting the efficiency of Agrobacterium mediated transformation were optimized, such as the threshold concentration of kanamycin for selection, the pre-culture time of the explants, the suitable infection time of the Agrobacterium, the working concentration of the Agrobacterium and the best working concentration of acetosyringone (AS). Using this protocol, atnhx1-cDNA was transfermed into A. melilotoides Pall and C.bus L successfully. 3 T₀ generation transgenic strains of A melilotoides Pall and 2 transgenic strains of C.bus L and some of kanamycin-resistant calli were obtained. The PCR, Southern blotting and RT-PCR identification of the T₀ transgenic plants show that the atnhx1-cDNA has already integrated into the genome of the transgenic plants and the target gene can express at the level of RNA transcription.Both the transgenic calli and the wild-type calli of A. melilotoides Pall and C.bus L were respectively cultured on the media containing different concentration of NaCl in order to test their resistance to NaCl. The results demonstrated that relative growth rates of the transgenic calli were higher than that of the wild-type calli.After being cultured respectively on the media containing different concentration of NaCl, the accumulations of free proline ,K⁺ and Na⁺ in the cells of both the transgenic calli and the wild-type calli of A. melilotoides Pall and C.bus L were investigated. The results showed that both the concentration of free proline and K⁺/Na⁺ in the transgenic calli were higher than that in wild-type calli under the same stress condition. Therefore, the intergration of atnhxl gene enhanced the resistance to the salt stress of the transgenic calli.In order to investigate whether the atnhxl gene transformation can enhance the salt tolerance of the T₀ transgenic plants, the K⁺ and Na⁺ concentration and relative conductivity of the transgenic plant leaves of A. melilotoides Pall and C.bus L were estimated when they were cultured under the stress triggered by various concentration of NaCl from 0 to 250mmol/L. The results indicated that the K⁺/Na⁺ rate of the transgenic plants was higher than that of wild-type plant under the same stress condition, but the situation of relative conductivity was on the opposite. Therefore, the transfer of atnhx1 gene can also enhance the salt tolerance of the target plant.According to the multiply alignment of amino acid sequences of several plants Betaine Aldehyde Dehydrogenase(BADH) from NCBI, a pair of degenerate primers were designed against the conserved regions that was used for RT-PCR to amplify a 728bp cDNA segment from Hordeum vulgare L.var.nudum Hook.f. Based on the sequence of this cDNA segment, primers were designed for 3' RACE, 5'RACE and RT-PCR. Then a 538bp cDNA segment (the sequence of 3' end of badh) was acquired by 3' RACE. Finally by RT-PCR, a 1, 657bp cDNA fragment was cloned from Hordeum vulgare L.var.nudum Hook.f. This cDNA fragment contained a 1,512bp ORF coding the putative Betaine Aldehyde Dehydrogenase(BADH) and comprising 503 amino acid residues. Compared by blast, this putative Betaine Aldehyde Dehydrogenase was high homologue to other plants' BADH, and the highest homology to bbd2, a badh gene from barely, was 98.4%. The result indicates the gene cloned from Hordeum vulgare L.var.nudum Hook.f is a BADH gene named hvbadh1, and its Genbank accession number is EF492983. Expressing hvbadhl in the E.coli protein fusion and purification system, a new fusion protein about 96.3KD, composed by hvBADH1 (about 54.2KD) and MBP (about 42KD), was found by SDS-PAGE.In order to construct an expression vector which can express hvbadhl in plants, an expression cassette, including a CaMV35S promoter, hvbadhl and a NOS polyA sequence, was introduced into the T-DNA region of the binary vector pCAMBIA1301. The plant selectable marker gene of the vector is hptII delivering hygromycin B resistance to the transgenic plants. This recombined binary vector named as pCAM-ba was transfermed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, which could be used for plant transformation directly.2 regenerated T₀ transgenic plants of Nicotiana tabacum Qinyan95 with hygromycin B resistance were obtained via Agrobacterium tumefaciens-mediated transformation and characterized by PCR, and RT-PCR. The PCR and RT-PCR analysis of the T₀ generation transgenic plants show that the hvbadh1 gene has already integrated into the genome of the transgenic plants and the target gene can express at the level of RNA transcription.