蒺藜苜蓿MtNSN1的克隆与功能分析

doi:10.11686/cyxb2019145

草业学报 ›› 2019, Vol. 28 ›› Issue (8): 200-208.DOI: 10.11686/cyxb2019145

蒺藜苜蓿MtNSN1的克隆与功能分析

张智琦, 王珍, 张铁军, 龙瑞才, 杨青川, 康俊梅^*

中国农业科学院北京畜牧兽医研究所,北京100193

收稿日期:2019-03-05 出版日期:2019-08-20 发布日期:2019-08-20
通讯作者: *,E-mail: kangjunmei@caas.cn
作者简介:张智琦(1993-),女,内蒙古包头人,硕士。E-mail: suixi_suibuxi@126.com
基金资助:
国家科技部中欧政府间科技合作重点专项“牧草和豆类作物育种以提高欧盟和中国蛋白质自给”(2017YFE0111000)和中国农业科学院北京畜牧兽医研究所科技创新工程特设项目(ASTIP-IAS-TS-14)资助

Cloning and functional analysis of the MtNSN1 gene in Medicago truncatula

ZHANG Zhi-qi, WANG Zhen, ZHANG Tie-jun, LONG Rui-cai, YANG Qing-chuan, KANG Jun-mei^*

Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2019-03-05 Online:2019-08-20 Published:2019-08-20
Contact: *,E-mail: kangjunmei@caas.cn

摘要/Abstract

摘要： 核干因子(NS)是一类广泛存在于动物细胞核中的GTP结合蛋白,主要参与胚的形成和细胞增殖,对维持细胞周期具有重要作用。本研究旨在初探蒺藜苜蓿MtNSN1的表达模式及其对生长发育的调控功能。克隆获得蒺藜苜蓿MtNSN1基因cDNA全长2193 bp,其中开放阅读框为1800 bp,编码599个氨基酸,蛋白分子量133.7 kDa。进化树分析显示MtNSN1与大豆NSN1的氨基酸同源性为66.67%。组织器官表达分析表明,MtNSN1在蒺藜苜蓿花中表达量最高,根次之,叶中表达量最低。RNA原位杂交的结果显示该基因主要在蒺藜苜蓿生长发育活跃的茎尖分生组织及其周围的幼嫩叶中表达。MtNSN1转化野生型拟南芥得到超表达植株,通过统计分析表明超表达植株的主根长度和叶片数量都明显优于野生型。在超表达植株中5个细胞周期标记基因的表达水平均比野生型拟南芥显著上调;利用DNA化学诱变剂博来霉素对植株进行处理,发现超表达植株的根系受抑制程度小于野生型。以上结果表明,MtNSN1与蒺藜苜蓿茎尖分生组织的维持及地上、地下器官的发育有关,同时异源表达MtNSN1在转录水平上影响拟南芥细胞周期的表达。

关键词: 蒺藜苜蓿, MtNSN1, 分生组织, 细胞周期

Abstract: Nucleostemin (NS), a nucleolar GTP-binding protein widely present in animals, is involved in embryogenesis and cell proliferation, which are heavily dependent on cell cycling. This research investigated the expression pattern of MtNSN1 and the function in plant growth and development in Medicago truncatula. The full-length cDNA of MtNSN1 (2193 bp) was cloned revealing an open reading frame of 1800 bp encoding 599 amino acids of molecular weight 133.7 kDa. Phylogenetic analysis showed that MtNSN1 is related to NSN1 in Glycine max with a homology of 66.67%. The highest transcript level of MtNSN1 was detected in flowers, followed by roots, and the lowest in leaves. In situ hybridization showed that was mainly expressed in the shoot apical meristem and the nearby leaf primordia and emerging leaves. Transgenic plants overexpressing MtNSN1 had longer primary roots, and the transgenic plants possessed more leaves than the non-transgenic ones. Real-time quantitative PCR analysis indicated that five cell cycling marker genes were significantly up-regulated in plants overexpressing MtNSN1. Treatment with the genotoxic agent bleomycin demonstrated that the length of the primary roots of plants overexpressing MtNSN1 was longer than that of the non-transgenic control plants. These findings suggest that MtNSN1 may be involved in the maintenance of the shoot of Medicago truncatula, which is crucial for the development of both underground and aerial organs. In addition, ectopic expression of MtNSN1 altered the transcriptional level of the cell cycling genes.

Key words: Medicago truncatula, MtNSN1, meristem, cell cycle

张智琦, 王珍, 张铁军, 龙瑞才, 杨青川, 康俊梅. 蒺藜苜蓿MtNSN1的克隆与功能分析[J]. 草业学报, 2019, 28(8): 200-208.

ZHANG Zhi-qi, WANG Zhen, ZHANG Tie-jun, LONG Rui-cai, YANG Qing-chuan, KANG Jun-mei. Cloning and functional analysis of the MtNSN1 gene in Medicago truncatula[J]. Acta Prataculturae Sinica, 2019, 28(8): 200-208.

参考文献

[1] Beekman C, Nichane M, De Clercq S, et al. Evolutionarily conserved role of nucleostemin: Controlling proliferation of stem/progenitor cells during early vertebrate development. Molecular and Cellular Biology, 2006, 26(24): 9291-9301.
[2] Ma H, Pederson T.Depletion of the nucleolar protein nucleostemin causes G1 cell cycle arrest via the p53 pathway. Molecular and Cellular Biology, 2007, 18(7): 2630-2635.
[3] Jérôme A, Charles B, Michel C T.The cell cycle of early mammalian embryos: Lessons from genetic mouse models. Cell Cycle, 2006, 5(5): 499-502.
[4] Britton R A.Role of GTPases in bacterial ribosome assembly. Annual Review of Microbiology, 2009, 63: 155-176.
[5] Tsai R T, Mckay R D.A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes & Development, 2002, 16(23): 2991-3003.
[6] Mudgil Y, Shiu S H, Stone S L, et al. A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family. Plant Physiology, 2004, 134(1): 59-66.
[7] Shao L H, Zheng X W, Li C.Cloning and expression analysis of a U-Box gene of E3 ubiquitin ligase from Medicago truncatula. Acta Prataculturae Sinica, 2016, 25(7): 62-72.
邵麟惠, 郑兴卫, 李聪. 蒺藜苜蓿E3泛素连接酶U-box基因克隆及表达分析. 草业学报, 2016, 25(7): 62-72.
[8] Zhu Q, Meng L, Hsu J K, et al. GNL3L stabilizes the TRF1 complex and promotes mitotic transition. Journal of Cell Biology, 2009, 185(5): 827-839.
[9] Wang X, Xie B, Zhu M, et al. Nucleostemin-like 1 is required for embryogenesis and leaf development in Arabidopsis. Plant Molecular Biology, 2012, 78(1/2): 31-44.
[10] Fang X, Fan Y, Liu Z, et al. Nucleostemin mRNA is expressed in both normal and malignant renal tissues. European Urology Supplements, 2006, 5(2): 30.
[11] Wang X, Gingrich D K, Deng Y, et al. A nucleostemin-like GTPase required for normal apical and floral meristem development in Arabidopsis. Molecular and Cellular Biology, 2012, 23(8): 1446-1456.
[12] Jeon Y, Park Y J, Cho H K, et al. The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis. Journal of Experimental Botany, 2015, 66(20): 6297-6310.
[13] Dai M S, Sun X X, Lu H.Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2. Molecular and Cellular Biology, 2008, 28(13): 4365-4376.
[14] Cho H K, Ahn C S, Lee H S, et al. Pescadillo plays an essential role in plant cell growth and survival by modulating ribosome biogenesis. Plant Journal, 2013, 76(3): 393-405.
[15] Eshed Y, Baum S F, Perea J V, et al. Establishment of polarity in lateral organs of plants. Current Biology, 2001, 11(16): 1251-1260.
[16] Wang Z, Wang X, Xie B, et al. Arabidopsis nucleostemin-like 1 (NSN1) regulates cell cycling potentially by cooperating with nucleosome assembly protein AtNAP1;1. BMC Plant Biology, 2018, 18(1): 99.
[17] Sitaraman J, Bui M, Liu Z.LEUNIG_HOMOLOG and LEUNIG perform partially redundant functions during Arabidopsis embryo and floral development. Plant Physiology, 2008, 147(2): 672-681.
[18] She C W, Song Y C.Genomic homology between Arabidopsis thaliana and distantly related plants revealed by comparative genomic in situ hybridization with Arabidopsis DNA probe. Chinese Journal of Biochemistry and Molecular Biology, 2007, 23(11): 946-952.
佘朝文, 宋运淳. 拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性. 中国生物化学与分子生物学报, 2007, 23(11): 946-952.
[19] Li J, Yu M, Bing J, et al. The application of RNA in situ hybridization technique in maize. Plant Physiology Journal, 2015, 51(12): 2280-2286.
李洁, 于明, 邴杰, 等. RNA原位杂交技术在玉米研究中的应用. 植物生理学报, 2015, 51(12): 2280-2286.
[20] Li Z Y.Molecular cloning and functional analysis of MsHSP70 gene in Medicago sativa L. Beijing: Chinese Academy of Agricultural Sciences, 2015.
栗振义. 紫花苜蓿热激蛋白基因MsHSP70的克隆及功能分析. 北京: 中国农业科学院, 2015.
[21] Gao J.Functional analysis of histone methyltransferase (SDG25) and nucleosome assemble protein1 (NAP1) in Arabidopsis. Shanghai: Fudan University, 2012.
高娟. 拟南芥组蛋白甲基转移酶SDG25和组蛋白分子伴侣NAP1的功能研究. 上海: 复旦大学, 2012.
[22] Culligan K M, Robertson C E, Foreman J, et al. ATR and ATM play both distinct and additive roles in response to ionizing radiation. Plant Journal, 2006, 48(6): 947-961.
[23] Daigle D M, Laura R, Berghuis A M, et al. YjeQ, an essential, conserved, uncharacterized protein from Escherichia coli, is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics. Biochemistry, 2002, 41(37): 11109-11117.
[24] Sridhar V V, Anandkumar S, Deyarina G, et al. Transcriptional repression of target genes by LEUNIG and SEUSS, two interacting regulatory proteins for Arabidopsis flower development. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(31): 11494-11499.
[25] Jibran R, Tahir J, Cooney J, et al. Arabidopsis AGAMOUS regulates sepal senescence by driving jasmonate production. Frontiers in Plant Science, 2017, 8: 2101.
[26] Lingjun M, Tao L, Guang P, et al. Nucleostemin deletion reveals an essential mechanism that maintains the genomic stability of stem and progenitor cells. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(28): 11415-11420.
[27] Donati G, Montanaro L, Derenzini M.Ribosome biogenesis and control of cell proliferation: p53 is not alone. Cancer Research, 2012, 72(7): 1602-1607.
[28] Meng L, Lin T, Tsai R Y.Nucleoplasmic mobilization of nucleostemin stabilizes MDM2 and promotes G2-M progression and cell survival. Journal of Cell Science, 2008, 121(24): 4037-4046.
[29] Waadt R, Schmidt L K, Lohse M, et al. Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant Journal, 2008, 56(3): 505-516.

蒺藜苜蓿MtNSN1的克隆与功能分析

Cloning and functional analysis of the MtNSN1 gene in Medicago truncatula

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