桑树Na+/H+逆向转运蛋白基因(MnNHX 1)的克隆与耐盐力表达

doi:10.11707/j.1001-7488.20150803

摘要/Abstract

摘要：

[目的] 研究川桑液泡膜型Na⁺/H⁺逆向转运蛋白(NHX)基因的功能,探究桑树耐盐机制,为植物抗逆基因工程筛选提供优良的候选基因。[方法] 以川桑基因组数据库为基础,基于同源基因序列的保守性,以川桑叶片cDNA为模板克隆川桑液泡膜型NHX基因; 利用生物学软件和在线公共平台,分析所得基因编码的蛋白质序列及功能结构域,并构建系统进化树,分析与其他物种的亲缘关系。采用荧光定量PCR方法研究在NaCl胁迫条件下不同时间段'湖桑32号'根、茎、叶等不同组织中桑树NHX 1表达量的变化情况; 通过构建超量表达载体,将其转化到拟南芥中,分析转基因拟南芥在NaCl胁迫环境中的种子发芽数,根长、侧根生长情况和幼苗成活率,并对转基因拟南芥连续浇灌含高浓度NaCl的营养液,研究过量表达NHX基因对拟南芥的影响。[结果] 本研究得到1个液泡膜型NHX基因,命名为MnNHX 1(GenBank登录号: KJ720637); 该基因ORF长度为1 644 bp,编码547个氨基酸残基,具有Na⁺/H⁺交换泵, 且在其上游含有抑制剂氨氯吡嗪脒结合位点(LFFIYLLPPI)以及糖基化位点等结构域,TMHMM在线程序预测MnNHX1具有12个明显的跨膜结构区; 系统进化树分析结果显示,MnNHX1具有较高的保守性,先与源于蔷薇科的桃聚合,与桑树形态学和基因组进化分析分类结果一致。荧光定量PCR试验表明,在无NaCl胁迫条件下桑树NHX 1 在‘湖桑32号’根、茎、叶中均有表达; 在NaCl胁迫处理12 h后,根、茎中桑树NHX 1的表达量显著增加,而后回落; 而在胁迫处理24 h后,叶中桑树NHX 1 的表达量显著提高,随后回落。过量表达MnNHX 1 的转基因拟南芥在NaCl胁迫环境中,种子发芽率低于野生型,而根长和侧根生长情况以及幼苗成活率都优于野生型; 连续浇灌含高浓度NaCl营养液的转基因拟南芥生长状态更为优良。[结论] MnNHX 1为优良的植物耐盐基因,在桑树中为组成型表达,并受NaCl胁迫诱导,表现出组织特异性。过量表达MnNHX 1的拟南芥耐盐能力显著提高,生存在盐胁迫环境中,依然具有良好的生长和发育能力。

关键词: 桑树, Na⁺/H⁺逆向转运蛋白, 非生物胁迫, 耐盐性, 基因功能

Abstract:

[Objective] To study the function of Na⁺/H⁺antiporter (NHX) in vacuolar membrane from mulberry tree Morus notabilis, and to explore the mechanism of salt tolerance in mulberry, and to provide an excellent candidate gene for the screening of plant resistance gene engineering. [Method] In this study, a Na⁺/H⁺ antiporter gene named as MnNHX 1 was identified based on the M. notabilis genomic database and other homologous sequences. The MnNHX 1 was cloned using the cDNA from M. notabilis leaves as template. The analysis of the primary structure and functional domains from MnNHX1 was completed by the bioinformatics analysis. The phylogenetic tree was generated to analyse the relationships between mulberry NHX 1 and other species. Quantitative PCR was conducted to analyse the expression profiles of mulberry NHX 1 in different tissues of M.multicaulis ‘Husang No.32’ and treatment time under NaCl stress. The overexpression vector was constructed and transformed into Arabidopsis thaliana. The seed germination rate, the growth of roots and the survival rate of seedlings of the transgenic A. thaliana were analyzed under NaCl stress. Furthermore, the transgenic A. thaliana was continuously irrigated with the nutrient solution containing high concentration of NaCl to study the functional effects of MnNHX 1 gene in the transgenic A. thaliana. [Result] We cloned a Na⁺/H⁺ antiporter gene designated as MnNHX 1 (GenBank accession No. KJ720637). The open reading frame (ORF) of MnNHX 1 is 1 644 bp and encodes a protein of 547 amino acid with a Na⁺/H⁺ exchange pump. At the upstream of this pump, there are some domains such as inhibitors amiloride binding sites (LFFIYLLPPI) and glycosylation sites. The analysis of the online program of TMHMM showed that MnNHX1 have 12 obvious transmembrane region. Phylogenetic analysis showed that MnNHX 1 was firstly clustered with Prunus persica from the Rosaceae family, which is consistent with morphological classification and genomic phylogenetic analysis of mulberry. Quantitative PCR showed that expression of mulberry NHX 1 was detected in roots, stems and leaves of M.multicaulis ‘Husang No. 32’ without NaCl treatment. The expression levels of mulberry NHX 1 were significantly increased followed by a drop in roots and stems after 12 h salt treatment, and in leaves after 24 h treatment. The seed germination rate of transgenetic A. thaliana overexpressed MnNHX 1 was lower than that of the wild plants under the salt condition, but root length, growth of lateral roots and survival rate of seedlings were higher than those of the wild plants. When the transgenetic A.thaliana seedlings irrigated with high concentration NaCl, they grew better than the wide type plants. [Conclusion] MnNHX 1 is a excellent candidate gene for improving the salt tolerance and can be constitutively expressed in mulberry tree. However, its induced expression pattern showed tissue specificity under salt condition. Furthermore, overexpression of MnNHX 1 in A. thaliana can significantly improve the salt tolerance of the transgenic A. thaliana.

Key words: Morus, Na⁺/H⁺ antiporter, abiotic stress, salt tolerance, gene function

中图分类号:

S718.46

边晨凯, 龙定沛, 刘雪琴, 魏从进, 龚加红, 赵爱春. 桑树Na⁺/H⁺逆向转运蛋白基因(MnNHX 1)的克隆与耐盐力表达[J]. 林业科学, 2015, 51(8): 16-25.

Bian Chenkai, Long Dingpei, Liu Xueqin, Wei Congjin, Gong Jiahong, Zhao Aichun. Cloning and Expression to Salt Stress of Na⁺/H⁺ Antiporter Gene (MnNHX 1) in Mulberry Tree[J]. Scientia Silvae Sinicae, 2015, 51(8): 16-25.

参考文献

刘雪琴, 丁天龙, 魏从进,等. 2014. 13 份桑树杂交组合F1代的耐盐性和耐旱性鉴定. 蚕业科学, 40(5): 764-773. (Liu X Q, Ding T L, Wei C J, et al. 2014. An evaluation on salt and drought tolerance of F₁ generations from 13 mulberry hybrid combinations. Science of Sericulture, 40(5): 764-773.[in Chinese])
秦俭, 何宁佳, 黄先智,等. 2010. 桑树生态产业与蚕丝业的发展. 蚕业科学, 36(6): 984-989. (Qin J, He N J, Huang X Z,et al. 2010. Development of mulberry ecological industry and sericulture. Science of Sericulture, 36(6): 984-989.[in Chinese])
唐欣, 王瑞辉, 杨秀艳, 等. 2014.唐古特白刺液泡膜Na⁺/H⁺逆向运输蛋白基因NtNHX 1 的克隆与表达分析. 林业科学, 50(3):38-44. (Tang X, Wang R H, Yang XY,et al. 2014. Isolation and expression analysis of a vacuolar membrane N⁺/H⁺ antiporter gene NtNHX 1 from Nitraria tangutorum. Scientia Silvae Sinicae, 50(3):38-44.[in Chinese])
张雨良, 张智俊, 杨峰山,等. 2009. 新疆盐生植物车前PmNHX 1 基因的克隆及生物信息学分析. 中国生物工程杂志, 29(1):27-33. (Zhang Y L, Zhang Z J, Yang F S,et al. 2009.Cloning and bioinformatics analysis of PmNHX 1 gene from Xinjiang halophyte Plantago maritima. China Biotechnology, 29(1):27-33.[in Chinese])
张智俊, 杨洋, 罗淑萍, 等. 2011. 毛竹液泡膜Na⁺/H⁺ 逆向运输蛋白基因克隆及表达分析. 农业生物技术学报, 19(1):69-76. (Zhang Z J, Yang Y, Luo S P,et al. 2011.Molecular cloning and expression analysis of a vacuolar Na⁺/H⁺ antiporter gene in Moso bamboo. Journal of Agricultural Biotechnology, 19(1):69-76.[in Chinese])
An B Y, Luo Y, Li J R,et al. 2008. Expression of a vacuolar Na⁺/H⁺ antiporter gene of alfalfa enhances salinity tolerance in transgenic Arabidopsis. Acta Agron Sin, 34(4):557-564.
Almansouri M, Kinet J M, Lutts S. 2001. Effect of salt and osmotic stresses on germination in durum wheat (Triticum durum Desf.). Plant and Soil, 231(2):243-254.
Apse M P, Aharon G S, Snedden W A, et al. 1999. Salt tolerance conferred by overexpression of a vacuolar Na⁺/H⁺antiporter in Arabidopsis. Science, 285(5431):1256-1258.
Apse M P, Sottosanto J B, Blumwald E. 2003. Vacuolar cation/H⁺exchange, ion homeostasis, and leaf development are altered in a TDNA insertional mutant of AtNHX 1 ,the Arabidopsis vacuolar Na⁺/H⁺ antiporter. Plant Journal, 36(2):229-239.
Bassil E, Coku A, Blumwald E. 2012. Cellular ion homeostasis: emerging roles of intracellular NHX Na⁺/H⁺antiporters in plant growth and development. Journal of Experimental Botany, 63(16):5727-5740.
Bassil E, Ohto M, Esumi T,et al. 2011. The Arabidopsis intracellular Na⁺/H⁺ antiporters NHX5 and NHX6 are endosome associated and necessary for plant growth and development. The Plant Cell Online, 23(1): 224-239.
Blumwald E. 2000. Sodium transport and salt tolerance in plants. Current Opinion in Cell Biology, 12(4): 431-434.
Brini F, Masmoudi K. 2012. Ion transporters and abiotic stress tolerance in plants. ISRN Molecular Biology, 2012: 1-13.
Chen L H, Zhang B, Xu Z Q. 2008. Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na⁺ /H⁺ antiporter gene AtNHX 1 in common buckwheat (Fagopyrum esculentum). Transgenic Research, 17(1):121-132.
Fukuda A, Nakamur A, Tanaka Y. 1999. Molecular cloning and expression of the Na⁺/H⁺exchanger gene in Oryza sativa. Biochin Biophys Acta, 1446(12):149-155.
Gaxiola R A, Li J, Undurraga S,et al. 2001. Drought and salt tolerant plants result from overexpression of the AVP1 H⁺pump. Proceedings of the National Academy of Sciences, 98(20):11444-11449.
Guan B, Hu Y, Zeng Y,et al. 2011. Molecular characterization and functional analysis of a vacuolar Na⁺/H⁺ antiporter gene (HcNHX 1) from Halostachys caspica. Molecular Biology Reports, 38(3):1889-1899.
He N, Zhang C, Qi X,et al. 2013. Draft genome sequence of the mulberry tree Morus notabilis. Nature Communications, 4. doi:10.1038/ncomms3445.
Leidi E O, Barragán V, Rubio L, et al. 2010. The AtNHX 1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato. Plant Journal, 61(3):495-506.
Liu P, Yang G D, Li H,et al. 2010. Overexpression of NHX 1 s in transgenic Arabidopsis enhances photoprotection capacity in high salinity and drought conditions. Acta Physiol Plant, 32(1):81-90.
McDowell J M, Huang S, McKinney E C, et al. 1996. Structure and evolution of the actin gene family in Arabidopsis thaliana. Genetics, 142(2):587-602.
Munns R, Tester M. 2008. Mechanisms of salinity tolerance. Annu Rev Plant Biol, 59:651-681.
Qiao W H, Zhao X Y, Li W,et al. 2007. Overexpression of AeNHX 1 , a root-specific vacuolar Na⁺/H⁺ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants. Plant Cell Rep, 26(9): 1663-1672.
Qiu Q S. 2012. Plant and yeast NHX antiporters: Roles in membrane trafficking. Journal of Integrative Plant Biology,54(2):66-72.
Quintero F J, Blatt M R, Pardo J M. 2000. Functional conservation between yeast and plant endosomal Na⁺/H⁺ antiporters. FEBS Letters, 471(2):224-228.
Sagarika M, Hemasundar A,Byeong-ha L, et al. 2014. Cloning and functional characterization of a vacuolar Na⁺/H⁺ antiporter gene from mungbean (VrNHX 1 ) and its ectopic expression enhanced salt tolerance in Arabidopsis thaliana. PloS One, 9(10): e106678.
Saleki R,Young P G, Lefebvre D D,et al. 1993. Mutants of Arabidopsis thaliana capable of germination under saline conditions. Plant Physiol, 101(3):839-845.
Shabala S, Cuin T A. 2008. Potassium transport and plant salt tolerance. Physiol Plant, 133 (4):651-669.
Sun Y, Wang D, Bai Y, et al. 2006. Studies on the overexpression of the soybean GmNHX 1 in Lotus corniculatus:The reduced Na⁺level is the basis of the increased salt tolerance. Chinese Science Bulletin, 51(11):1306-1315.
The Angiosperm Phylogeny Group. 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society, 161(2):105-121.
Vasekina A V, Yershovp V, Reshetova O S,et al. 2005. Vacuolar Na⁺/H⁺ antiporter from barley: identification and response to salt stress. Biochemistry-Moscow, 70(1):100-107.
Verslues P E, Agarwal M, Katiyar-Agarwal S, et al. 2006. Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. The Plant Journal, 45(4): 523-539.
Wei C, Liu X, Long D,et al. 2014. Molecular cloning and expression analysis of mulberry MAPK gene family. Plant Physiology and Biochemistry, 77: 108-116.
Wu C, Gao X, Kong X,et al. 2009. Molecular cloning and functional analysis of a Na⁺/H⁺ antiporter gene ThNHX 1 from a halophytic plant Thellungiella halophila. Plant Mol Biol Rep, 27(1):1-12.
Wu C A, Yang G D, Meng Q W,et al. 2004. The cotton GhNHX 1 gene encoding a novel putative tonoplast Na⁺/H⁺ antiporter plays an important role in salt stress. Plant and Cell Physiology, 45(5):600-607.
Zhang H, Liu Y, Xu Y,et al. 2012. A newly isolated Na⁺/H⁺ antiporter gene, DmNHX 1 , confers salt tolerance when expressed transiently in Nicotiana benthamiana or stably in Arabidopsis thaliana. Plant Cell Tiss Organ Cult, 110(2): 189-200.
Zhang H X, Blumwald E. 2001. Tranagenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nature Biotehnology, 19(8):765-768.
Zhu J K. 2001. Plant salt tolerance. Trends in Plant Science, 6(2):66-71.
Zoerb C, Noll A, Karl S,et al. 2005. Molecular characterization of Na⁺/H⁺antiporter(ZmNHX) of maize (Zea mays L.) and their expression under salt stress. Journal of Plant Physiology, 162(1):55-66.

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