Abstract：Gastrodia elata is a traditional Chinese medicinal herb that can be used for both medicine and food. G. elata has no roots or leaves, and can only obtain nutrients through the digestion of infected Armillaria mellea. Nitrogen nutrition is essential for the growth of G. elata and A.mellea, and glutamine synthetase (GS) plays an important role in regulating nitrogen metabolism. Previous studies have shown that overexpression of the cytoplasmic GS1 gene can improve plant nitrogen utilization, plant development, and salt stress tolerance, but the resistance of the GS1 gene to low temperature stress is still unclear. In this study, the 5' CDS of GS1 gene was screened from the transcriptome database of G. elata, and it's full length was cloned by Nested-PCR. The overexpression vector pH2GW7.0-35s-GS1 and prokaryotic expression vector pET-32a-GS1 were constructed. pET-32a-GS1 was transformed into Escherichia coli Bl21 for protein express and purification to obtain GS1 enzyme protein. The optimal pH of GS1 enzyme was 4 and the optimal temperature was 50 ℃. Under low concentration of 5 mmol/L Ca2+, Mg2+ and K+ had a promoting effect on GS1 enzyme activity. While at high concentration of 9 mmol/L metal ions had a inhibitory effect on GS1 enzyme activity. Compared with wild A. mellea, overexpression of GS1 significantly enhanced the expression levels of GS, glutamate synthase (GoGAT), glutamate dehydrogenase (GDH), glutathione reductase (GR) and glutathione peroxidase (GPX) genes related to glutamate and glutathione synthesis under low temperature conditions of 13 ℃, increased the content of proline, glutathione and soluble sugar, and reduced the content of H2O2 and malondialdehyde (MDA), significantly promoted the growth of A. mellea. These results indicated that overexpression of G. elata GS1 gene could enhance the growth ability of A. mellea under low temperature conditions. This study provids a reference for further research on enhancing growth and yield of G. elata under low temperature.
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