|其他摘要||Ammonium is one of the most important nitrogen sources for plants. An appropriate concentration of NH4+ promotes plant growth, while causes toxicity with excess ammonium. Salt stress often imposes an adverse effect on nitrogen metabolism and plant growth. Salicornia europaea, one of the most salt-tolerant halophyte, often grows at salt lake side where soil is saline, humid and anaerobic. Not only the growth of this species is promoted by NaCl, it also has strong ability in NH4+ absorption and ammonium tolerance. However, it is still unclear about its nitrogen metabolism characteristics under saline environment, the molecular mechanism of NH4+ transport, and why NaCl can enhance its ammonium tolerance. In this study, S. europaea was tested under various Na+/NH4+ treatment to uncover the characteristics of ions transport and stress tolerance. Six members of ammonium transporter (AMT) gene family were cloned, and analyzed with gene expression characteristics, and gene functions were identified. In addition, we selected suitable RT-qPCR reference genes for S. europaea under various conditions and found a simple method for further assembly of targeted unigenes in a transcriptome. The main results were as follows:
(1) Na+ accumulation and improvement of nitrogen assimilation enzyme activity could be the main reasons for NaCl facilitated S. europaea growth. Cell size became larger under NaCl (< 200 mM) treatment, which could be the cause that Na+ accumulation improved turgor pressure by osmotic regulation. Furthermore, NaCl promoted corresponding genes expression levels and enzymes activity for nitrogen or ammonium assimilation, such as glutamine synthetase (GS) and glutamate dehydrogenase (GDH). NH4+ concentration was decreased and toxicity reduced by NaCl treatment in root under ammonium stress, which indicate NaCl also improved S. europaea ammonium tolerance.
(2) Through data statistics of researches on screening plant reference genes in the last ten years, and combining the transcriptome expression profile data of S. europaea, we found that none of the analyzed genes can guarantee universally stable expressions. The tested genes belonging to multigene family could be one reason of variations in the published studies. Therefore, we validated suitable reference genes for gene expression analysis in S. europaea under various conditions (salt stress, nitrogen/ ammonium stress, drought stress, cold stress, heat stress, various tissues, various age) by RT-qPCR, which will benefit future accurate gene quantitative in S. europaea.
(3) In order to find the molecular mechanism of NH4+ transport, we cloned six SeAMT genes from S. europaea after obtain longer sequence using a method for further assembly of AMT unigenes in transcriptome. These genes were expressed in different parts of the plant, to complete the NH4+ absorption from rhizosphere and NH4+ transport in the body. SeAMT1.2a, SeAMT1.2b and SeAMT3.1 were mainly expressed in the root, in charge of the NH4+ absorption from rhizosphere root cells. SeAMT1.1 and SeAMT1.3 mainly were expressed in shoot for NH4+ transport and storage in overground part of plant. The expression quantity of SeAMT1.4 was very low, which could be detected both in root and shoot. Confronted with nitrogen deficiency, expression of SeAMT1.2a and SeAMT1.2b increased with time to adapt to the nitrogen starvation adversity. Six SeAMT genes were all induced up-regulation by high concentration of NH4+, and then feedback inhibition. SeAMT genes were also regulated by NaCl. The analysis of GFP subcellular localization indicated that nearly all SeAMT protein orientation on the cell membrane, supporting the conclusion that SeAMTs were membrane transporters of NH4+. Through complementary function test in yeast 31019b and Arabidopsis thaliana qko, we found that six SeAMT have different ability for NH4+ transport. Members of SeAMT1 subfamily undertook almost all tasks for ammonium transport. Although SeAMT3.1 was expressed in root, it did not showed any ability of ammonium transport in heterologous expression systems. SeAMT1.2a and SeAMT1.2b, with high sequence similarity, had more powerful ability for NH4+ transport than other SeAMT genes. They were expressed in root and were induced up-regulation by nitrogen starvation. It indicated that SeAMT1.2a and SeAMT1.2b were two important genes for ammonium transport and nitrogen metabolism regulation. In addition, we found the two genes also improved salt resistance of yeast and A. thaliana. According to the result of gene transformation in yeast CY162, SeAMT1.2a promoted K+ transport under a low K+ condition. This could be one of reasons that the gene improve salt resistance of genetically modified organisms|