陆地棉转录因子GhNAC7的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2017.03.002

摘要/Abstract

摘要： 【【目的】从陆地棉中克隆GhNAC7，分析其结构和功能，研究其在棉花不同组织中以及叶片不同发育时期的表达量。并转入拟南芥进一步探究其在棉花叶片衰老过程中的作用。【方法】利用中国农业科学院棉花研究所棉花生物学国家重点实验室建立的棉花衰老叶片cDNA文库中的序列，获得1个含有NAM结构域的EST，使用Oligo 6.71设计引物，重新在陆地棉叶片cDNA中进行克隆。使用Gene Structure Display Server软件分析GhNAC7结构，使用在线工具PlantCARE分析启动子序列，利用在线工具GenScan进行氨基酸序列翻译。同时，利用拟南芥基因组数据库（TAIR）进行序列比对，选取得分较高的NAC家族基因，使用MEGA 6.06软件和GeneDoc软件进行进化树分析和氨基酸比对。以XbaⅠ和SacⅠ为酶切位点构建35S::GhNAC7-GFP融合表达载体，分析其在洋葱表皮细胞中的瞬时表达，进行亚细胞定位。利用实时荧光定量PCR技术分析GhNAC7在棉花不同组织、不同叶片发育时期以及在200 μmol·L^-1 ABA调控下的表达量。通过构建pGhNAC7-GUS融合表达载体并转拟南芥，分析其启动子特异性。以EcoRⅠ和SalⅠ为酶切位点，利用pBI101和pBI121载体，分别构建融合表达载体并转拟南芥进行过表达分析。【结果】从陆地棉中成功克隆GhNAC7，其全长为1 064 bp，包含3个外显子，2个内含子。生物信息学分析结果表明，GhNAC7开放阅读框为834 bp，可编码277个氨基酸，其蛋白质分子量为31.35 kD，等电点为9.22。结构域分析表明其属于NAC转录因子的NAM亚家族，进化树分析显示GhNAC7与ANAC041、ANAC083同源性最高，其中，GhNAC7与ANAC083结构域位置均为17—58 aa。其启动子核心元件包含一系列与衰老、激素、胁迫相关的顺式作用元件。亚细胞定位表明其蛋白为核蛋白。组织特异性表明GhNAC7在真叶、子叶、花、花药和衰老真叶中均明显表达，其中在衰老的真叶中表达量最高。启动子特异性分析表明，其GUS活性在衰老的叶片中最强。在拟南芥中过表达该基因，转基因植株比野生型表现出明显的衰老症状。荧光定量PCR分析表明，ABA处理后6 h GhNAC7明显上调表达，并在48 h表达量达到最高，这表明ABA可调控GhNAC7表达从而调节棉花叶片衰老。【结论】GhNAC7可以促进棉花叶片衰老并受ABA的调控。

关键词: 陆地棉, NAM结构域, 叶片衰老, GhNAC7, 过表达

Abstract: 【Objective】The primary objectives of this experiment are to clone GhNAC7 gene, analyze its structure, detect its expression in different tissues of cotton and at different developmental leaf senescence stages. Furthermore, its function in cotton leaf senescence was further studied through transforming GhNAC7 gene into Arabidopsis. 【Method】Based on the cotton senescent leaves cDNA library, which was built by state key laboratory of cotton biology of institute of cotton research of CAAS, this gene was cloned from upland cotton using an expressed sequence tag (EST) containing NAM domain after designing primer using Oligo 6.71. Gene Structure Display Sever was conducted to analyze its structure, PlantCARE was used on-line to study its promoter sequence, and GenScan was simultaneously performed to translate amino acid on-line. Meanwhile, NAC family genes with higher scores were chosen after aligning sequence from Arabidopsis in TAIR. Afterwards MEGA 6.06 was used to display evolutionary relationships of the gene and GeneDOC was conducted to perform sequence alignment of amino acids. Via constructing 35S::GhNAC7-GFP fusion expression vector with XbaⅠand SacⅠrestriction sites, subcellular localization of GhNAC7 was studied by transient expression analysis of onion epidermal cells. Expression profiles of GhNAC7 in various tissues, in response to 200 μmol·L^-1 ABA treatment and developmental leaf senescence stages were investigated through quantitative real-time PCR (qRT-PCR). Its promoter specificity was conducted by transforming into Arabidopsis thaliana after constructing pGhNAC7-GUS fusion expression vector. Simultaneously, using pBI101 and pBI121 with EcoRⅠand SalⅠrestriction sites were used to construct fusion expression vectors, and then over-expression analysis was performed by transforming GhNAC7 into Arabidopsis thaliana.【Result】 In this study, a novel gene GhNAC7 was successfully cloned from upland cotton (Gossypium hirsutum L.). Its full-length was 1 064 bp with three exons and two introns. Results of bioinformatics analysis exhibited that its open reading frame (ORF) was 834 bp, which encoding 277 amino acids. The molecular weights of GhNAC7 encoding protein were 31.35 kD and isoelectric point was 9.22. Domain analysis of GhNAC7 showed that it belongs to the NAM subgroup of NAC family, moreover phylogenetic tree analysis showed that GhNAC7 has the closest genetic relationship with ANAC041 and ANAC083, and GhNAC7 displayed the same domain positions with ANAC083 at 17-58 aa. Its core promoter elements were also predicted, which contained a series of aging, hormone, stress-related cis-acting elements. Subcellular localization elucidated the protein of GhNAC7 encoding is a nucleoprotein. Tissue-specific analysis showed that this gene was significantly expressed in true leaves, cotyledons, flowers, anthers and senescent leaves, but displayed the highest expression in senescent leaves. And in promoter specificity analysis, it was also exhibited the strongest GUS activity in senescent leaf. Over-expression in transgenic Arabidopsis verified evident symptoms of aging compared to the wild type. Fluorescence quantitative PCR analysis showed that the gene expression was significantly up-regulated after 6 h of ABA treatment and displayed the highest at 48 h, so it was hypothesized that ABA could regulate GhNAC7 gene expression to mediate cotton leaf senescence.【Conclusion】It was concluded that GhNAC7 gene could promote cotton leaf senescence and be regulated by ABA.

Key words: upland cotton, NAM domain, leaf senescence, GhNAC7, over-expression

郑学伟，SHAH Syed Tariq，范术丽，魏恒玲，庞朝友，李鸿彬，喻树迅. 陆地棉转录因子GhNAC7的克隆及功能分析[J]. 中国农业科学, 2017, 50(3): 426-436.

ZHENG XueWei, SHAH Syed Tariq, FAN ShuLi, WEI HengLing, PANG ChaoYou, LI HongBin, YU ShuXun. Molecular Cloning and Functional Analysis of GhNAC7 in Upland Cotton (Gossypium hirsutum L.)[J]. Scientia Agricultura Sinica, 2017, 50(3): 426-436.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.03.002

https://www.chinaagrisci.com/CN/Y2017/V50/I3/426

参考文献

[1] Gunapati S, Naresh R, Ranjan S, RANJAN S, NIGAM D, HANS A, VERMA P C, GADRE R, PATHRE U V, SANE A P, SANE V A. Expression of GhNAC2 from G. herbaceum, improves root growth and imparts tolerance to drought in transgenic cotton and Arabidopsis. Scientific reports, 2016, 6: 24978.

[2] Wang J Y, Wang J P, Yang H F. Identification and functional characterization of the NAC gene promoter from Populus euphratica. Planta, 2016, 244(2): 417-427.

[3] Rahman H, Ramanathan V, Nallathambi J, DURAIALAGARAJA S, MUTHURAJAN R. Over-expression of a NAC67 transcription factor from finger millet (Eleusine coracana L.) confers tolerance against salinity and drought stress in rice. BMC biotechnology, 2016, 16(1): 7.

[4] Shah S T, Pang C, Hussain A, FAN S, SONG M, ZAMIR R, YU S. Molecular cloning and functional analysis of NAC family genes associated with leaf senescence and stresses in Gossypium hirsutum L.. Plant Cell, Tissue and Organ Culture, 2014, 117(2): 167-186.

[5] Kim H J, Nam H G, Lim P O. Regulatory network of NAC transcription factors in leaf senescence. Current Opinion in Plant Biology, 2016, 33: 48-56.

[6] Souer E, van Houwelingen A, Kloos D, MOL J, KOES R. The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell, 1996, 85(2): 159-170.

[7] Jin J, Zhang H, Kong L, GAO G, LUO J. PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic acids research42(Database issue): D1182., 2014,

[8] 康桂娟, 曾日中, 聂智毅, 黎瑜, 代龙军, 段翠芳. 植物NAC转录因子的研究进展. 生物技术通报, 2012, 28(11): 21-26.

Kang G J, Zeng R Z, Nie Z Y, LI Y, DAI L J, DUAN C F. Research progress of plant NAC transcription factors. Biotechnology Bulletin, 2012, 28(11): 21-26. (in Chinese)

[9] 陈娜, 蒋晶, 曹必好, 雷建军，陈长明. 植物NAC转录因子功能研究新进展. 分子植物育种, 2015, 13(6): 1407-1414.

Chen N, Jiang J, Cao B H, LEI J J, CHEN C M. The latest progresses on plant NAC transcription factors function. Molecular Plant Breeding, 2015, 13(6): 1407-1414. (in Chinese)

[10] Fan K, Bibi N, Gan S,LI F, YUAN S, NI M, WANG M, SHEN H, WANG X. A novel NAP member GhNAP is involved in leaf senescence in Gossypium hirsutum. Journal of experimental botany, 2015, 66(15): 4669-4682.

[11] Evans O, Dou L, Guo Y, PANG C, WEI H, SONG M, FAN S, YU S. GhNAC18, a novel cotton (Gossypium hirsutum L.) NAC gene, is involved in leaf senescence and diverse stress responses. African Journal of Biotechnology, 2016, 15(24): 1233-1245.

[12] He X, Zhu L, Xu L, GUO W, ZHANG X. GhATAF1, a NAC transcription factor, confers abiotic and biotic stress responses by regulating phytohormonal signaling networks. Plant Cell Reports, 2016, 35(10): 2167-2179.

[13] Wang G, Zhang S, Ma X, WANG Y, KONG F, MENG Q. A stress-associated NAC transcription factor (SlNAC35) from tomato plays a positive role in biotic and abiotic stresses. Physiologia plantarum, 2016, 158(1): 45-64.

[14] Niu F, Wang C, Yan J, GUO X, WU F, YANG B, DEYHOLOS M K, JIANG Y. Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcriptional activator modulating reactive oxygen species accumulation and cell death. Plant Molecular Biology, 2016, 92(1): 89-104.

[15] Christiansen M W, Matthewman C, Podzimska-Sroka D, O’SHEA C, LINDEMOSE S, Møllegaard N E, HOLME L B, HEBELSTRUP K, SKRIVER K, GREGERSEN P L. Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence. Journal of Experimental Botany, 2016, 67(17): 5259-5273.

[16] Nakashima K, Tran L S P, Van Nguyen D, FUJITA M, MARUYAMA K, TODAKA D, LTO Y, HAYASHI N, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress‐responsive gene expression in rice. The Plant Journal, 2007, 51(4): 617-630.

[17] Hu H, You J, Fang Y, ZHU X, QI Z, XIONG L. Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice. Plant molecular biology, 2008, 67(1/2): 169-181.

[18] Oh S K, Lee S, Yu S H, CHOI D. Expression of a novel NAC domain-containing transcription factor (CaNAC1) is preferentially associated with incompatible interactions between chili pepper and pathogens. Planta, 2005, 222(5): 876-887.

[19] Xie Q, Frugis G, Colgan D, CHUA N H. Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes & development, 2000, 14(23): 3024-3036.

[20] 宋国立, 崔荣霞, 王坤波, 郭立平, 黎绍惠, 王春英, 张香娣. 改良CTAB法快速提取棉花DNA. 棉花学报, 1998, 10(5): 273-275.

Song G L, Cui R X, Wang K B, GUO L P, LI S H, WANG C Y, ZHANG X D. A rapid improved CTAB method for extraction of cotton genomic DNA. Cotton Science, 1998, 10(5): 273-275. (in Chinese)

[21] Wu A, Liu J. An improved method of chromosome walking for promoter sequences cloning. Chinese journal of biochemistry and molecular biology, 2005, 22(3): 243-246.

[22] Guo A Y, Zhu Q H, Chen X, Luo J C. GSDS: a gene structure display server. Yi Chuan, 2007, 29(8): 1023-1026.

[23] Lichtenthaler H K. [34] Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in enzymology, 1987, 148: 350-382.

[24] 张文香, 范术丽, 宋美珍, 庞朝友, 魏恒玲, 喻树迅. 棉花GhMADS29启动子克隆及表达分析. 棉花学报, 2015, 25(4): 309-315.

Zhang W X, Fan S L, Song M Z, PANG C Y, WEI H L, YU S X. Cloning and expression analysis of the promoter of GhMADS29 from cotton. Cotton Science, 2015, 25(4): 309-315. (in Chinese)

[25] Meng C, Cai C, Zhang T, GUO W. Characterization of six novel NAC genes and their responses to abiotic stresses in Gossypium hirsutum L.. Plant Science, 2009, 176(3): 352-359.

[26] He X J, Mu R L, Cao W H, ZHANG Z G, ZHANG J S, CHEN S Y. AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. The Plant Journal, 2005, 44(6): 903-916.

[27] Zhao F, Ma J, Li L, FAN S, GUO Y, SONG M, WEI H, PANG C. GhNAC12, a neutral candidate gene, leads to early aging in cotton (Gossypium hirsutum L.). Gene, 2016, 576(1): 268-274.

[28] Oda-Yamamizo C, Mitsuda N, Sakamoto S, OGAWA D, OHME-TAKAGI M, OHMIYA A. The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves. Scientific reports, 2016, 6: 23609.

[29] GAO S, Gao J, Zhu X, SONG Y, LI Z, REN G, ZHOU X, KUAI B. ABF2, ABF3 and ABF4 promote ABA-mediated chlorophyll degradation and leaf senescence by transcriptional activation of chlorophyll catabolic genes and senescence-associated genes in Arabidopsis. Molecular Plant, 2016, 9(9): 1272-1285.

[30] Shah S T, Pang C, Fan S, SONG M, ARAIN S, YU S. Isolation and expression profiling of GhNAC transcription factor genes in cotton (Gossypium hirsutum L.) during leaf senescence and in response to stresses. Gene, 2013, 531(2): 220-234.

[31] Yang S D, Seo P J, Yoon H K, PARK C M. The Arabidopsis NAC transcription factor VNI2 integrates abscisic acid signals into leaf senescence via the COR/RD genes^[W]. The Plant Cell, 2011, 23(6): 2155-2168.

[32] Pimenta M R, Silva P A, Mendes G C, ALVES J R, CAETANO H D N, MACHADO J P B, BRUSTOLINI O J B, CARPINETTI P A, MELO B P, SILVA J C F, ROSADO G L, FERREIRA M F S, DAL-BIANCO M, PICOLI E A d T, ARAGAO F J L, RAMOS H J O, FONTES E P B. The stress-induced soybean NAC transcription factor GmNAC81 plays a positive role in developmentally programmed leaf senescence. Plant and Cell Physiology, 2016, 57(5): 1098-1114.

[33] Mahmood K, El-Kereamy A, Kim S H, nambara e, rothstein s j. ANAC032 positively regulates age-dependent and stress-induced senescence in Arabidopsis thaliana. Plant and Cell Physiology, 2016: pcw120.