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Scientia Silvae Sinicae ›› 2015, Vol. 51 ›› Issue (8): 16-25.doi: 10.11707/j.1001-7488.20150803

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Cloning and Expression to Salt Stress of Na+/H+ Antiporter Gene (MnNHX 1) in Mulberry Tree

Bian Chenkai, Long Dingpei, Liu Xueqin, Wei Congjin, Gong Jiahong, Zhao Aichun   

  1. State Key Laboratory of Silkworm Genome Biology Key Laboratory for Sericulture Functional Genomics and Biotechnology of Ministry of Agriculture Southwest University Chongqing 400716
  • Received:2014-10-08 Revised:2015-01-21 Online:2015-08-25 Published:2015-09-10

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Abstract:

[Objective] To study the function of Na+/H+ antiporter (NHX) in vacuolar membrane from mulberry tree Morus notabilis, and to explore the mechanism of salt tolerance in mulberry, and to provide an excellent candidate gene for the screening of plant resistance gene engineering. [Method] In this study, a Na+/H+ antiporter gene named as MnNHX 1 was identified based on the M. notabilis genomic database and other homologous sequences. The MnNHX 1 was cloned using the cDNA from M. notabilis leaves as template. The analysis of the primary structure and functional domains from MnNHX1 was completed by the bioinformatics analysis. The phylogenetic tree was generated to analyse the relationships between mulberry NHX 1 and other species. Quantitative PCR was conducted to analyse the expression profiles of mulberry NHX 1 in different tissues of M.multicaulis ‘Husang No.32’ and treatment time under NaCl stress. The overexpression vector was constructed and transformed into Arabidopsis thaliana. The seed germination rate, the growth of roots and the survival rate of seedlings of the transgenic A. thaliana were analyzed under NaCl stress. Furthermore, the transgenic A. thaliana was continuously irrigated with the nutrient solution containing high concentration of NaCl to study the functional effects of MnNHX 1 gene in the transgenic A. thaliana. [Result] We cloned a Na+/H+ antiporter gene designated as MnNHX 1 (GenBank accession No. KJ720637). The open reading frame (ORF) of MnNHX 1 is 1 644 bp and encodes a protein of 547 amino acid with a Na+/H+ exchange pump. At the upstream of this pump, there are some domains such as inhibitors amiloride binding sites (LFFIYLLPPI) and glycosylation sites. The analysis of the online program of TMHMM showed that MnNHX1 have 12 obvious transmembrane region. Phylogenetic analysis showed that MnNHX 1 was firstly clustered with Prunus persica from the Rosaceae family, which is consistent with morphological classification and genomic phylogenetic analysis of mulberry. Quantitative PCR showed that expression of mulberry NHX 1 was detected in roots, stems and leaves of M.multicaulis ‘Husang No. 32’ without NaCl treatment. The expression levels of mulberry NHX 1 were significantly increased followed by a drop in roots and stems after 12 h salt treatment, and in leaves after 24 h treatment. The seed germination rate of transgenetic A. thaliana overexpressed MnNHX 1 was lower than that of the wild plants under the salt condition, but root length, growth of lateral roots and survival rate of seedlings were higher than those of the wild plants. When the transgenetic A.thaliana seedlings irrigated with high concentration NaCl, they grew better than the wide type plants. [Conclusion] MnNHX 1 is a excellent candidate gene for improving the salt tolerance and can be constitutively expressed in mulberry tree. However, its induced expression pattern showed tissue specificity under salt condition. Furthermore, overexpression of MnNHX 1 in A. thaliana can significantly improve the salt tolerance of the transgenic A. thaliana.

Key words: Morus, Na+/H+ antiporter, abiotic stress, salt tolerance, gene function

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