关键词: 杨树, Na⁺/H⁺反向运输蛋白, 反转录PCR, 盐诱导的差异表达

Abstract:

The Na⁺/H⁺ antiporter is a ubiquitous membrane protein gene family of bacteria, plants and mammals. In Arabidopsis thaliana, six NHX isoforms have been identified to date and some of which have a major function in response to NaCl. The Populus NHX₁ and NHX₆ cDNA isolation was performed by underlying the electronic cloning technique, based on the Populus EST databases and P. trichocarpa genome sequence using AtNHX₁ and AtNHX₆ nucleotide cDNA sequence information. Two cDNA clones encoding NHX were isolated from the cDNA prepared from cambial zone of Populus tomentosa by the RT-PCR method. These two cDNAs are 1 635 bp and 1 709 bp in length with corresponding open reading frames (ORFs) which are capable of encoding the protein of 544 and 526 amino acids (aa), respectively. The deduced aa sequence of the PtNHX₁ and PtNHX₆ (GenBank accession number AY660749 and AY832912) proteins shares 79%,76%,69%, 74% and 82%,82%, 27%,26% identity with the other recently isolated A. thaliana, Oryza sativa, Triticum aestivum and Zea mays NHX₁ and NHX₆, respectively. When poplar genomic DNA was digested with selected restriction enzymes and then analysed on a Southern blot, we found one to four bands that were labelled, suggesting a low-copy gene (or a few highly homologous genes) within the poplar genome. Tissue differential expression indicated that PtNHX₁ and PtNHX₆ transcripts have their mRNA products in roots, stems and leaves, but have some differences in PtNHX₁ and PtNHX₆ gene. As for PtNHX₁, it was expressed predominantly in roots, cambium, immature xylem, young leaf and mature leaf, with some expression in the phloem and mature xylem zone of the stem. The PtNHX₆ transcripts are the most abundant mRNA products in roots, young and mature leaf, and a low-abundance was detected in phloem, cambium and xylem. To confirm the possible regulation of PtNHX₁ and PtNHX₆ gene expression by NaCl, the RT-PCR detection of the expression abundance of the PtNHX₁ and PtNHX₆ genes was performed at different NaCl concentration (from 100 to 600 mmol·L^-1) after exposure to the salt treatment. The results showed that PtNHX₁ and PtNHX₆ message levels were increased for the NaCl concentration of up to 400 mmol·L^-1 but declined with the concentration of more than 400 mmol·L^-1 and this expression pattern matched the ABA content detected in the leaves. The data suggest that PtNHX₁ and PtNHX₆ are induced by salt stress.

Key words: Populus, Na⁺/H⁺ antiporters, RT-PCR, Differential expression induced by salt stress