摘要
由焦磷酸(PPi)驱动的膜整合焦磷酸酶(PPase)在生物适应逆境方面可能起着重要作用,包括H^+-PPase、Na^+-PPase和Na^+,H^+-PPase三个亚家族。相比于H^+-PPase,其它两个亚家族PPase近年来才在原核生物(特别是动物肠道菌)中被发现,有关它们的应用研究更是匮乏。通过两轮巢式PCR从人肠道宏基因组中扩增得到甲基戊糖梭菌膜整合焦磷酸酶(CmPP)全长基因。序列分析表明该基因含有一个完整的ORF(长2 100 bp,编码一个699 aa的蛋白质,G+C含量为59.9%,碱基分布均衡)。膜拓扑学结构预测CmPP是一个由16个跨膜α螺旋组成的膜蛋白,与目前已鉴定过的膜PPase跨膜结构相符。序列比对分析推测CmPP属于K+依赖型Na^+,H^+-PPase亚家族成员。另外,通过农杆菌转化获得了CmPP转基因烟草。对其子一代植株的抗旱分析发现,在干旱胁迫条件下CmPP转基因株系的干物质含量、总叶绿素含量和根系活力均下降,而其叶片MDA含量和电解质渗透率均升高,但相同胁迫下的变化程度都显著低于野生型烟草。这些结果表明CmPP基因的导入能够有效提高烟草的抗旱能力。
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 twoPPase subfamilies were recently detected only in prokaryotes（especially animal gut microbial）,certainly with scarce application researches. Inthis 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 Cm PP gene contained an entire ORF of 2100 bp encoding aprotein of 699 aa,with the G＋C percentage of 59.9 % and balanced base distribution. Topological prediction showed Cm PP as a multi-spanningmembrane protein consisting of 16 transmembrane α-helix segments,in accordance with the membrane topological model of those knownmembrane PPases. Furthermore,sequence alignment analysis implicated Cm PP as a K~＋-dependent member of Na＋,H＋-PPase subfamily. Inaddition,we obtained the Cm PP transgenic tobacco plants through Agrobacterium transformation. Under drought stress conditions,the F1 plants of Cm PP transgenic lines had decreased values in dry matter content,total chlorophyll content and root activity,but increased valuesin leaf MDA content and electrolyte permeability. However,all these changes in transgenic lines were significantly lower than those in wildtype tobacco under the same stress. Conclusively,our results indicated that introduction of the Cm PP gene efficiently improved the droughtresistance in tobacco.
出处
《生物技术通报》
CAS
CSCD
北大核心
2017年第6期81-88,共8页
Biotechnology Bulletin
基金
国家自然科学基金项目(31160169
31460067)
关键词
甲基戊糖梭菌
膜整合焦磷酸酶
基因克隆
转基因烟草
抗旱性
Clostridium methylpentosum
membrane-bound pyrophosphatase
gene cloning
transgenic tobacco
drought resistance
