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拟南芥叶绿体分裂突变体x17-3和pd50的基因鉴定与分析

李劲宇 高原 张琴 刘小敏 高宏波

李劲宇, 高原, 张琴, 刘小敏, 高宏波. 拟南芥叶绿体分裂突变体x17-3和pd50的基因鉴定与分析[J]. 北京林业大学学报, 2018, 40(4): 86-95. doi: 10.13332/j.1000-1522.20170433
引用本文: 李劲宇, 高原, 张琴, 刘小敏, 高宏波. 拟南芥叶绿体分裂突变体x17-3和pd50的基因鉴定与分析[J]. 北京林业大学学报, 2018, 40(4): 86-95. doi: 10.13332/j.1000-1522.20170433
Li Jinyu, Gao Yuan, Zhang Qin, Liu Xiaomin, Gao Hongbo. Genetic identification and analysis of chloroplast division mutants x17-3 and pd50 in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2018, 40(4): 86-95. doi: 10.13332/j.1000-1522.20170433
Citation: Li Jinyu, Gao Yuan, Zhang Qin, Liu Xiaomin, Gao Hongbo. Genetic identification and analysis of chloroplast division mutants x17-3 and pd50 in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2018, 40(4): 86-95. doi: 10.13332/j.1000-1522.20170433

拟南芥叶绿体分裂突变体x17-3和pd50的基因鉴定与分析

doi: 10.13332/j.1000-1522.20170433
基金项目: 

国家自然科学基金项目 31501090

详细信息
    作者简介:

    李劲宇,博士生。主要研究方向:植物分子生物学。Email:lijinyu2009@hotmail.com 地址:100083北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    刘小敏,讲师。主要研究方向:植物分子生物学。Email: liuxiaomin@bjfu.edu.cn 地址:同上

    高宏波,教授,博士生导师。主要研究方向:植物分子生物学。Email:gaobjfu@yahoo.com 地址:同上

  • 中图分类号: Q943.2

Genetic identification and analysis of chloroplast division mutants x17-3 and pd50 in Arabidopsis thaliana

  • 摘要: 目的叶绿体起源于内共生的蓝细菌,它通过与细菌类似的分裂方式进行增殖以维持遗传稳定。叶绿体分裂需要起源于原核和真核细胞的蛋白高度协调。拟南芥是研究叶绿体分裂的模式植物。在过去的20年中,人们对拟南芥叶绿体分裂蛋白复合体进行了初步的研究。然而, CRL基因在叶绿体分裂中的功能还不清楚。方法本研究通过突变体筛选和图位克隆鉴定得到2个新的crl突变体,分别为x17-3和pd50。通过显微观察和分子生物学方法分析了x17-3和pd50中叶绿体分裂表型、CRL基因剪接方式和mRNA含量以及叶绿素含量。最后通过转化互补和RNA干扰(RNAi)技术进一步确认了基因功能。结果x17-3和pd50的叶绿体形态与野生型相比有较大差异,表现为叶绿体体积增大,细胞中叶绿体数量减少。x17-3叶绿体数量为野生型的40%,而pd50叶绿体减少到只有1~4个,植物生长也受到明显抑制。通过粗定位及测序分析发现x17-3和pd50的CRL基因存在突变,突变位点在内含子并且影响mRNA剪接,最终导致阅读框移码突变。通过实时荧光定量PCR分析发现,pd50中CRL mRNA含量比野生型和x17-3明显降低。遗传互补实验进一步验证了x17-3和pd50中叶绿体分裂和植物生长抑制表型是CRL基因突变导致的。应用RNAi技术抑制CRL基因表达也能产生明显的叶绿体分裂异常表型。此外,pd50和x17-3的叶绿素含量比野生型明显降低。结论本项工作为进一步揭示CRL基因的功能提供新的研究材料和实验依据。

     

  • 图  1  x17-3和pd50突变体的叶绿体分裂和植物生长表型分析

    A.生长4周的拟南芥叶肉细胞叶绿体的形态,标尺为10 μm;B.每个叶肉细胞叶绿体数量与叶肉细胞平面面积之间的关系;C.生长4周的拟南芥植物形态,标尺为1 cm。

    Figure  1.  Phenotype analysis of the chloroplast division and plant growth in x17-3 and pd50 mutant

    A, morphology of chloroplasts from true leaf of four-week-old Arabidopsis thaliana plants, scale bar is 10 μm; B, relationship between chloroplast number per mesophyll cell and mesophyll cell plan area of four-week-old plants; C, plant morphology of four-week-old plants, scale bar is 1 cm.

    图  2  x17-3突变体的基因粗定位与测序分析

    A. 30个x17-3 F2单株与4个分子标记的连锁情况,*表示杂合的植物;B. CRL和分子标记在5号染色体上的位置;C.CRL基因结构及x17-3和pd50突变位点,黑色矩形和直线分别代表外显子和内含子,星号表示突变体中CRL基因突变位点。

    Figure  2.  Rough mapping and sequencing analysis of x17-3

    A, 4 molecular markers linked to the CRL locus and used for the identification of recombinants in 30 x17-3 mutant individuals from a F2 mapping population, * indicates heterozygous plants; B, genetic linkage map of CRL gene and the molecular markers on chromosome 5; C, gene structure and the mutation site of CRL gene in x17-3 and pd50, black boxes and straight lines indicate exons and introns, respectively, * represents CRL mutation site of mutant.

    图  3  x17-3和pd50中CRL mRNA剪接分析

    A. WT、x17-3和pd50中CRL基因mRNA PCR扩增产物,M是DNA marker;B. x17-3中CRL mRNA序列分析,x17-3中增加的10 bp内含子导致蛋白质翻译发生移码,蛋白翻译提前终止,矩形和直线分别代表第8个和第9个外显子以及内含子,*代表终止密码子;C. pd50中CRL mRNA序列分析,pd50中CRL基因突变产生2种形式的mRNA,pd50(1)中缺失8 bp碱基,pd50(2)中保留了内含子,都导致蛋白质翻译发生移码,蛋白翻译提前终止,矩形和直线分别代表第1个和第2个外显子以及内含子,*代表终止密码子。

    Figure  3.  Splicing analysis of CRL mRNA in x17-3 and pd50

    A, PCR analysis of CRL mRNA in WT, x17-3 and pd50, M means DNA marke; B, analysis of CRL mRNA presenting in the x17-3 mutan, the extra 10 bp sequence included in x17-3 results in reading frame shift and early translational termination, the boxes and straight lines indicate the 8th, 9th exons and intron, respectively, * represents stop codon; C, analysis of CRL mRNA presenting in the pd50 mutant, the mutation in CRL gene in pd50 results in two isoform of CRL mRNA by alternative splicing, the 8 bp missing in pd50 (1) and intron retention in pd50 (2) result in reading frame shift and early translational termination, the boxes and straight lines indicate the 1st, 2nd exons and intron, respectively, * represents stop codon.

    图  4  x17-3和pd50中CRL mRNA含量的实时荧光定量PCR分析

    平均值±标准差,n=3;**表示在P < 0.01水平上差异显著(t-检验)。

    Figure  4.  Comparison of the mRNA level of CRL gene in x17-3 and pd50 by real-time qRT-PCR analysis

    mean±SD, n=3. ** refers to P < 0.01 by Student's t-test.

    图  5  x17-3和pd50转CRL基因互补表型分析

    A. WT、x17-3、pd50及x17-3、pd50转35S::CRL T2代植物叶绿体表型,标尺为10 μm;B. WT、x17-3、pd50及x17-3、pd50转35S::CRL T2代植物生长表型,标尺为1 cm;C.转基因拟南芥的PCR鉴定,M为DNA Marker,P为质粒对照。

    Figure  5.  Phenotypic analysis of chloroplasts and plant growth of x17-3 and pd50 complemented with a wild-type CRL transgene

    A, morphology of chloroplasts of x17-3 and pd50 complemented with a wild-type CRL transgene, scale bar is 10 μm; B, plant growth phenotypes of x17-3 and pd50 complemented with a wild-type CRL transgene; scale bar is 1 cm; C, PCR identification of x17-3 and pd50 transgenic plants, M means DNA marker, P means plasmid.

    图  6  CRL RNAi植物叶绿体表型和基因表达分析

    A. WT和CRL RNAi T2代植物叶绿体表型,标尺为10 μm;B. WT和CRL RNAi T2代植物生长表型,标尺为1 cm;C. CRL RNAi植物中CRL基因的半定量RT-PCR分析,三角形指示连续4倍梯度稀释cDNA模板的方向,PP2AA3基因为内参。

    Figure  6.  Phenotypic analysis of the chloroplasts and gene expression of CRL RNAi lines

    A, chloroplast morphology of CRL RNAi lines, scale bar is 10 μm; B, plant growth of CRL RNAi lines, scale bar is 1 cm; C, semi-quantitative RT-PCR analyses of CRL mRNA in different RNAi lines, the triangle indicates the four times serial dilution of cDNA template, PP2AA3 gene is used as the internal reference.

    图  7  WT、x17-3和pd50中光合色素含量分析

    平均值±标准差,n=4;**表示在P < 0.01水平上差异显著。

    Figure  7.  Analysis of photosynthetic pigments content of WT, x17-3 and pd50

    mean±SD, n=4. **refers to P < 0.01 by Student's t-test.

    表  1  本研究所用引物的序列

    Table  1.   Primers used in this study

    名称Name 序列Sequence
    CRL-4 5′- CTA CCA TGG GTA CCG AGT CGG GTT C -3′
    CRL-17 5′- CCT ACG CGT CTA GTC TTG CAA GAT GAG GGA C -3′
    CRL-7 5′- CTC ACT AAA TCC ACC ACT CGT AG -3′
    CRL-5 5′- CTA GTC TTG CAA GAT GAG GGA C -3′
    CRL-8 5′- CTT ACG CGT GTC TTG CAA GAT GAG GGA CC -3′
    CRL-RNAi1 5′- CCT CCA TGG GTA CCG AGT CGG G-3′
    CRL-RNAi2 5′- CTC AGA TCT GCT TGC TCC CTC GTA AAC TTC-3′
    CRL-RNAi3 5′- CCA ACG CGT ATG GGT ACC GAG TCG GG-3′
    CRL-RNAi4 5′- CCT AGA TCT CCG GTG AGA GAA CGA G-3′
    35S1 5′- CTG TCA CTT TAT TGT GAA GAT AGT GG -3′
    PP2AA3-1 5′- CCA AGC GGT TGT GGA GAA C -3′
    PP2AA3-2 5′- GAA CCA AAC ACA ATT CGT TGC TG -3′
    下载: 导出CSV

    表  2  突变体x17-3和pd50的遗传分析

    Table  2.   Genetic analysis of x17-3 and pd50

    杂交组合
    Cross combination
    总植株数
    Total plant number
    野生表型数
    WT plant number
    突变表型数
    Mutant plant number
    χ2
    x17-3×Ler 110 80 30 0.194
    pd50×Ler 131 103 28 0.735
    下载: 导出CSV
  • [1] Cavalier-Smith T. Membrane heredity and early chloroplast evolution[J]. Trends in Plant Science, 2000, 5(4):174-182. doi: 10.1016/S1360-1385(00)01598-3
    [2] Dyall S D, Brown M T, Johnson P J. Ancient invasions: from endosymbionts to organelles[J]. Science, 2004, 304:253-257. doi: 10.1126/science.1094884
    [3] Osteryoung K W, Pyke K A. Division and dynamic morphology of plastids[J]. Annual Review of Plant Biology, 2014, 65:443-472. doi: 10.1146/annurev-arplant-050213-035748
    [4] Chiang Y H, Zubo Y O, Tapken W, et al. Functional characterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development, growth, and division in Arabidopsis[J]. Plant Physiology, 2012, 160(1):332-348. doi: 10.1104/pp.112.198705
    [5] Okazaki K, Kabeya Y, Suzuki K, et al. The PLASTID DIVISION1 and 2 components of the chloroplast division machinery determine the rate of chloroplast division in land plant cell differentiation[J]. The Plant Cell, 2009, 21(6):1769-1780. doi: 10.1105/tpc.109.067785
    [6] Vercruyssen L, Tognetti V B, Gonzalez N, et al. GROWTH REGULATING FACTOR5 stimulates Arabidopsis chloroplast division, photosynthesis, and leaf longevity[J]. Plant Physiology, 2015, 167(3):817-832. doi: 10.1104/pp.114.256180
    [7] Gao Y, Liu H, An C, et al. Arabidopsis FRS4/CPD25 and FHY3/CPD45 work cooperatively to promote the expression of the chloroplast division gene ARC5 and chloroplast division[J]. The Plant Journal, 2013, 75(5):795-807. doi: 10.1111/tpj.2013.75.issue-5
    [8] Haswell E S, Meyerowitz E M. MscS-like proteins control plastid size and shape in Arabidopsis thaliana[J]. Current Biology, 2006, 16(1):1-11. doi: 10.1016/j.cub.2005.11.044
    [9] Veley K M, Marshburn S, Clure C E, et al. Mechanosensitive channels protect plastids from hypoosmotic stress during normal plant growth[J]. Current Biology, 2012, 22(5):408-413. doi: 10.1016/j.cub.2012.01.027
    [10] Raynaud C, Perennes C, Reuzeau C, et al. Cell and plastid division are coordinated through the prereplication factor AtCDT1[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(23):8216-8221. doi: 10.1073/pnas.0502564102
    [11] Wu G Z, Xue H W. Arabidopsis beta-ketoacyl-[acyl carrier protein] synthase I is crucial for fatty acid synthesis and plays a role in chloroplast division and embryo development[J]. The Plant Cell, 2010, 22(11):3726-3744. doi: 10.1105/tpc.110.075564
    [12] Nobusawa T, Umeda M. Very-long-chain fatty acids have an essential role in plastid division by controlling Z-ring formation in Arabidopsis thaliana[J]. Genes to Cells, 2012, 17(8):709-719. doi: 10.1111/j.1365-2443.2012.01619.x
    [13] Fan J, Zhai Z, Yan C, et al. Arabidopsis TRIGALACTOSYLDIACYLGLYCEROL5 interacts with TGD1, TGD2, and TGD4 to facilitate lipid transfer from the endoplasmic reticulum to plastids[J]. The Plant Cell, 2015, 27(10):2941-2955. https://www.ncbi.nlm.nih.gov/pubmed/26410300?dopt=Abstract
    [14] Xu C, Fan J, Cornish A J, et al. Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein[J]. The Plant Cell, 2008, 20(8):2190-2204. doi: 10.1105/tpc.108.061176
    [15] Fan J, Xu C. Genetic analysis of Arabidopsis mutants impaired in plastid lipid import reveals a role of membrane lipids in chloroplast division[J]. Plant Signaling & Behavior, 2011, 6(3):458-460. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_3142439
    [16] Okazaki K, Miyagishima S Y, Wada H. Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in Arabidopsis[J]. The Plant Cell, 2015, 27(3):663-674. doi: 10.1105/tpc.115.136234
    [17] 李劲宇, 安传敬, 刘小敏, 等.叶绿体分裂分子机制研究进展[J].植物生理学报, 2016, 52(11): 1733-1744. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zwslxtx201611021

    Li J Y, An C J, Liu X M, et al. Recent progress in the molecular mechanism of chloroplast division[J]. Plant Physiology Journal, 2016, 52(11): 1733-1744. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zwslxtx201611021
    [18] Asano T, Yoshioka Y, Kurei S, et al. A mutation of the CRUMPLED LEAF gene that encodes a protein localized in the outer envelope membrane of plastids affects the pattern of cell division, cell differentiation, and plastid division in Arabidopsis[J]. The Plant Journal, 2004, 38(3):448-459. doi: 10.1111/tpj.2004.38.issue-3
    [19] Simkova K, Kim C, Gacek K, et al. The chloroplast division mutant caa33 of Arabidopsis thaliana reveals the crucial impact of chloroplast homeostasis on stress acclimation and retrograde plastid-to-nucleus signaling[J]. The Plant Journal, 2012, 69(4):701-712. doi: 10.1111/tpj.2012.69.issue-4
    [20] Hudik E, Yoshioka Y, Domenichini S, et al. Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant[J]. Plant Physiology, 2014, 166(1):152-167. doi: 10.1104/pp.114.242628
    [21] Sugita C, Kato Y, Yoshioka Y, et al. CRUMPLED LEAF (CRL) homologs of Physcomitrella patens are involved in the complete separation of dividing plastids[J]. Plant & Cell Physiology, 2012, 53(6):1124-1133. https://www.ncbi.nlm.nih.gov/pubmed/22514088
    [22] Lichtenthaler H K. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes[J]. Methods in Enzymology, 1987, 148(1):350-382. doi: 10.1016-0076-6879(87)48036-1/
    [23] Porra R J, Thompson W A, Kriedemann P E. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy[J]. Biochimica et Biophysica Acta, 1989, 975(3):384-394. doi: 10.1016/S0005-2728(89)80347-0
    [24] Zhang X, Guo H. mRNA decay in plants: both quantity and quality matter[J]. Current Opinion in Plant Biology, 2017, 35:138-144. doi: 10.1016/j.pbi.2016.12.003
    [25] Luesse D R, Wilson M E, Haswell E S. RNA sequencing analysis of the msl2msl3, crl, and ggps1 mutants indicates that diverse sources of plastid dysfunction do not alter leaf morphology through a common signaling pathway[J/OL]. Frontiers in Plant Science, 2015, 6[2017-10-28]. https://doi.org/10.3389/fpls.2015.01148.
    [26] Reddy A S N, Barta A. Complexity of the alternative splicing landscape in plants[J]. The Plant Cell, 2013, 25(10):3657-3683. doi: 10.1105/tpc.113.117523
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  • 收稿日期:  2017-12-04
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