甲基戊糖梭菌膜整合焦磷酸酶提高转基因烟草的抗旱性

doi:10.13560/j.cnki.biotech.bull.1985.2016-1208

摘要/Abstract

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。

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

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

刘延娟, 杨玉梅, 刘娴, 高艳秀, 袁航, 龚明, 邹竹荣. 甲基戊糖梭菌膜整合焦磷酸酶提高转基因烟草的抗旱性[J]. 生物技术通报, 2017, 33(6): 81-88.

LIU Yan-juan, YANG Yu-mei, LIU Xian, GAO Yan-xiu, YUAN Hang, GONG Ming, ZOU Zhu-rong. Membrane-bound Pyrophosphatase of Clostridium methylpentosum Increases the Drought Resistance of Its Transgenic Tobacco[J]. Biotechnology Bulletin, 2017, 33(6): 81-88.

参考文献

[1] Baykov AA, Malinen AM, Luoto HH, et al. Pyrophosphate-fueled Na + and H + transport in prokaryotes[J]. Microbiol Mol Biol Rev, 2013, 77（2）：267-276.
[2] Holm NG, Baltscheffsky H. Links between hydrothermal environments, pyrophosphate, Na + , and early evolution[J]. Orig Life Evol Biosph, 2011, 41（5）：483-493.
[3] Serrano A, Pérez-Castiñeira JR, Baltscheffsky M, et al. H + -PPases：yesterday, today and tomorrow[J]. IUBMB Life, 2007, 59（2）：76-83.
[4] Tsai JY, Kellosalo J, Sun YJ, et al. Proton/sodium pumping pyrophosphatases：the last of the primary ion pumps[J]. Curr Opin Struct Biol, 2014, 27：38-47.
[5] Belogurov GA, Lahti R. A lysine substitute for K + . A460K mutation eliminates K + dependence in H + -pyrophosphatase of Carboxydothermus hydrogenoformans[J]. J Biol Chem, 2002, 277（51）：49651-49654.
[6] Gaxiola RA, Sanchez CA, Paez-Valencia J, et al. Genetic manipulation of a “vacuolar” H + -PPase：from salt tolerance to yield enhancement under phosphorus-deficient soils[J]. Plant Physiol, 2012, 159（1）：3-11.
[7] Schilling RK, Tester M, Marschner P, et al. AVP1：one protein, many roles[J]. Trends Plant Sci, 2017, 22（2）：154-162.
[8] Pasapula V, Shen G, Kuppu S, et al. Expression of an Arabidopsis vacuolar H + -pyrophosphatase gene（AVP1）in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions[J]. Plant Biotechnol, 2011, 9（1）：88-99.
[9] Wei A, He C, Li B, et al. The pyramid of transgenes TsVP and BetA effectively enhances the drought tolerance of maize plants[J]. Plant Biotechnol, 2011, 9（2）：216-229.
[10] Bao AK, Wang SM, Wu GQ, et al. Overexpression of the Arabidopsis H + -PPase enhanced resistance to salt and drought stress in transgenic alfalfa（Medicago sativa L. ）[J]. Plant Sci, 2009, 176（2）：232-240.
[11] Yang H, Knapp J, Koirala P, et al. Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorus-responsive type I H + -pyrophosphatase[J]. Plant Biotechnol, 2007, 5（6）：735-745.
[12] Paez-Valencia J, Sanchez-Lares J, Marsh E, et al. Enhanced proton translocating pyrophosphatase activity improves nitrogen use efficiency in Romaine lettuce[J]. Plant Physiol, 2013, 161（3）：1557-1569.
[13] Khoudi H, Maatar Y, Gouiaa S, et al. Transgenic tobacco plants expressing ectopically wheat H + -pyrophosphatase（H + -PPase）gene TaVP1 show enhanced accumulation and tolerance to cadmium[J]. J Plant Physiol, 2012, 169（1）：98-103.
[14] Luoto HH, Belogurov GA, Baykov AA, et al. Na + -translocating membrane pyrophosphatases are widespread in the microbial world and evolutionarily precede H + -translocating pyrophosphatases[J]. J Biol Chem, 2011, 24：21633-21642.
[15] Luoto HH, Baykov AA, Lahti R, et al. Membrane-integral pyrophosphatase subfamily capable of translocating both Na + and H + [J]. Proc Natl Acad Sci USA, 2013, 110（4）：1255-1260.
[16] Luoto HH, Nordbo E, Baykov AA, et al. Membrane Na + -pyrophosphatases can transport protons at low sodium concentrat-ions[J]. J Biol Chem, 2013, 49：35489-35499.
[17] 王学奎. 植物生理生化实验原理和技术[M]. 第2版. 北京：高等教育出版社, 2006.
[18] Guazzaroni ME, Morgante V, Mirete S, et al. Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment[J]. Environ Microbiol, 2013, 15（4）：1088-1102.
[19] Kapardar RK, Ranjan R, Grover A, et al. Identification and characterization of genes conferring salt tolerance to Escherichia coli from pond water metagenome[J]. Bioresour Technol, 2010, 101（11）：3917-3924.
[20] 刘畅, 罗著, 张梦如, 等. 蓝藻橙色类胡萝卜素蛋白的基因克隆及在大肠杆菌中的异源表达和功能分析[J]. 生物技术通报, 2016, 32（7）：138-145.
[21] Culligan EP, Marchesi JR, Hill C, et al. Mining the human gut microbiome for novel stress resistance genes[J]. Gut Microbes, 2012, 3（4）：394-397.
[22] Arumugam M, Raes J, Pelletier E, et al. Enterotypes of the human gut microbiome[J]. Nature, 2011, 473（7346）：174-180.
[23] Gill SR, Pop M, Deboy RT, et al. Metagenomic analysis of the human distal gut microbiome[J]. Science, 2006, 312（5778）：1355-1359.
[24] Hu H, Xiong L. Genetic engineering and breeding of drought-resistant crops[J]. Annu Rev Plant Biol, 2014, 65：715-741.
[25] Yang S, Vanderbeld B, Wan J, et al. Narrowing down the targets：towards successful genetic engineering of drought-tolerant crops[J]. Mol Plant, 2010, 3（3）：469-490.