木薯MeTPS9基因克隆及表达特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2017-0416

摘要/Abstract

摘要： 旨在揭示木薯MeTPS9基因在干旱、低温、遮荫等非生物胁迫应答中的作用。用RT-PCR的方法从木薯叶片中克隆MeTPS9基因（GenBank登录号：MF276889）,用MEGA软件构建系统进化树,用DnaSP软件分析基因结构变异,用PlantCARE分析启动子元件,用荧光定量PCR技术分析MeTPS9在不同组织中以及响应不同非生物胁迫的表达特性。结果显示,从木薯中克隆了一个TPS基因MeTPS9。该基因具有2 598 bp的开放阅读框,编码865个氨基酸,含有TPS家族保守结构域。系统进化树分析表明,MeTPS9与荠菜花和拟南芥中同源基因的亲缘关系较近,序列相似性分别为76.2%和75.6%。基因结构变异发现,木薯野生种和栽培种之间共有66个突变位点,其中11个为错义突变。启动子元件分析表明,MeTPS9含有干旱相关元件MBS、低温相关元件LTR、热胁迫相关元件HSE、以及ABA相关元件ABRE。实时荧光定量PCR分析表明,MeTPS9的表达量在须根中最高,在叶片中最低。而且,MeTPS9基因的表达能被干旱、低温、和遮荫处理显著诱导。MeTPS9在转录水平对木薯干旱、低温、和遮荫胁迫起调控作用,可作为候选基因进一步研究其在木薯抗逆中的作用。

关键词: 木薯, MeTPS9, 海藻糖合成酶, 结构变异, 表达分析, 非生物胁迫

Abstract: This work aims to reveal the function of MeTPS9 gene in abiotic stresses（e.g.,drought,cold,and shade）in cassava. RT-PCR method was applied to clone MeTPS9 gene（GenBank accession number：MF276889）from cassava leaves,MEGA software to construct its phylogenetic tree,DnaSP software to analysze its structural variation,PlantCARE software to analyze its promoter cis-element,and quantitative RT-PCR（qRT-PCR）to explore its expression patterns in different tissues and in response to different abiotic stresses. Results showed that a TPS（trehalose-6-phosphate synthase）gene,MeTPS9,was cloned from cassava. This gene had a 2598-bp open reading frame,encoded 865 amino acids,and contained a TPS conserved domain. Phylogenetic analysis revealed that MeTPS9 had close genetic relationship with its homologs from Capsella grandiflora and Arabidopsis thaliana,and the sequence similarity was up to 76.2% and 75.6%,respectively. Gene structural variation demonstrated that a total of 66 mutation sites were identified between wild and cultivated cassava species,of which 11 were mis-sensed. Promoter assay showed that MeTPS9 contained drought-related motif MBS,cold-related motif LTR,heat-related motif HSE,and ABA-related motif ABRE. qRT-PCR analysis revealed that MeTPS9 expressed the highest in fibrous root but the lowest in leaf. In addition,the expression of MeTPS9 was significantly induced by drought,cold,and shade stress. MeTPS9 played a regulatory role in abiotic stress such as drought,cold,and shade treatment at the transcriptional level and it could be served as a candidate to further study its functions in abiotic stress defense in cassava.

Key words: cassava, MeTPS9, trehalose-6-phosphate synthase, structure variation, expression analysis, abiotic stress

丁泽红, 付莉莉, 铁韦韦, 颜彦, 胡伟. 木薯MeTPS9基因克隆及表达特性分析[J]. 生物技术通报, 2017, 33(11): 84-91.

DING Ze-hong, FU Li-li, TIE Wei-wei, YAN Yan, HU Wei. Clone and Expression Characteristics of MeTPS9 Gene in Cassava[J]. Biotechnology Bulletin, 2017, 33(11): 84-91.

参考文献

[1] Okogbenin E, Setter TL, Ferguson M, et al. Phenotypic approaches to drought in cassava：review[J]. Frontiers in Physiology, 2013, 4（1）：93.
[2] 卢赛清, 盘欢, 马崇熙, 等. 2008年广西木薯低温冻害情况及应对措施[J]. 广西热带农业, 2009, 1（1）：21-22.
[3] Okoli PSO, Wilson GF. Response of cassava（Manihot esculenta Crantz）to shade under field conditions[J]. Field Crops Research, 1986, 14（4）：349-359.
[4] 郭蓓, 胡磊, 何欣, 等. 海藻糖-6-磷酸合成酶转基因烟草提高耐盐性的研究[J]. 植物学报, 2008, 25（1）：41-49.
[5] Garg AK, Kim JK, Owens TG, et al. Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses[J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99（25）：15898-15903.
[6] Li HW, Zang BS, Deng XW, et al. Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice[J]. Planta, 2011, 234（5）：1007-1018.
[7] Delorge I, Figueroa CM, Feil R, et al. Trehalose-6-phosphate synthase 1 is not the only active TPS in Arabidopsis thaliana[J]. Biochemical Journal, 2014, 466（2）：283-290.
[8] Kosmas SA, Argyrokastritis A, Loukas MG, et al. Isolation and characterization of drought-related trehalose 6-phosphate-synthase gene from cultivated cotton（Gossypium hirsutum L. ）[J]. Planta, 2006, 223（2）：329-339.
[9] 李莹, 柳参奎. 碱茅6-磷酸海藻糖合成酶基因的克隆及其耐逆性分析[J]. 草业学报, 2015, 24（1）：99-106.
[10] 丁菲, 庞磊, 李叶云, 等. 茶树海藻糖-6-磷酸合成酶基因（CsTPS）的克隆及表达分析[J]. 农业生物技术学报, 2012, 20（11）：1253-1261.
[11] 邢文婷, 李科明, 贾彩红, 等. 香蕉海藻糖合成酶基因（MaTPS5）生物学及表达特性分析[J]. 生物技术通报, 2017, 33（2）：118-124.
[12] 魏兵强, 王兰兰, 张茹, 等. 辣椒TPS家族成员的鉴定与CaTPS1的表达分析[J]. 园艺学报, 2016, 43（8）：1504-1512.
[13] 史健志, 徐燕, 纪德华, 等. 坛紫菜6-磷酸海藻糖合成酶（TPS）家族基因的克隆及表达特征分析[J]. 水产学报, 2015, 39（4）：485-495.
[14] Deng Y, Wang X, Guo H, et al. A trehalose-6-phosphate synthase gene from Saccharina japonica（Laminariales, Phaeophyceae）[J]. Molecular Biology Reports, 2014, 41（1）：529.
[15] Henry C, Bledsoe SW, Siekman A, et al. The trehalose pathway in maize：conservation and gene regulation in response to the diurnal cycle and extended darkness[J]. Journal of Experimental Botany, 2014, 65（20）：5959-5973.
[16] 丁泽红, 付莉莉, 铁韦韦, 等. 木薯MeNCED3基因克隆、结构变异及其表达分析[J]. 生物技术通报, 2016, 32（10）：148-153.
[17] Wang W, Feng B, Xiao J, et al. Cassava genome from a wild ancestor to cultivated varieties[J]. Nature Communications, 2014, 5（5）：5110.
[18] Yang HL, Liu YJ, Wang CL, et al. Molecular evolution of trehalose-6-phosphate synthase（TPS）gene family in populus, Arabidopsis and rice[J]. PLoS One, 2012, 7（8）：e42438.
[19] Hao GP, Zhang XH, Wang YQ, et al. Nucleotide variation in the NCED3 region of Arabidopsis thaliana and its association study with abscisic acid content under drought stress[J]. Journal of Integrative Plant Biology, 2009, 51（2）：175-183.
[20] Shinozaki K. Gene networks involved in drought stress response and tolerance[J]. Journal of Experimental Botany, 2007, 58（2）：221-227.