Abstract: Pyrophosphate（PPi）-energized membrane-bound pyrophosphatases（PPases）are composed of three subfamilies including H⁺-PPase,Na⁺-PPase and Na⁺,H⁺-PPase,and may play important roles in stress adaptation of organisms. In contrast to H⁺-PPase,other two PPase subfamilies were recently detected only in prokaryotes（especially animal gut microbial）,certainly with scarce application researches. In this study,through two rounds of nested PCR,we amplified the full-length gene of the membrane-bound PPase of Clostridium methylpentosum（CmPP）from human gut metagenome. As indicated by sequence analysis,the CmPP gene contained an entire ORF of 2100 bp encoding a protein of 699 aa,with the G+C percentage of 59.9 % and balanced base distribution. Topological prediction showed CmPP as a multi-spanning membrane protein consisting of 16 transmembrane α-helix segments,in accordance with the membrane topological model of those known membrane PPases. Furthermore,sequence alignment analysis implicated CmPP as a K⁺-dependent member of Na⁺,H⁺-PPase subfamily. In addition,we obtained the CmPP transgenic tobacco plants through Agrobacterium transformation. Under drought stress conditions,the F₁ plants of CmPP transgenic lines had decreased values in dry matter content,total chlorophyll content and root activity,but increased values in leaf MDA content and electrolyte permeability. However,all these changes in transgenic lines were significantly lower than those in wild-type tobacco under the same stress. Conclusively,our results indicated that introduction of the CmPP gene efficiently improved the drought resistance in tobacco.

Key words: Clostridium methylpentosum, membrane-bound pyrophosphatase, gene cloning, transgenic tobacco, drought resistance