MicroRNA828负调控缺磷胁迫诱导的番茄花青素生物合成

doi:10.3864/j.issn.0578-1752.2015.15.002

摘要/Abstract

摘要： 【目的】深入解析番茄miR828的生物学功能，特别是参与番茄缺磷胁迫响应和花青素生物合成的调控作用。【方法】分别利用psRNATarget和RLM-5′ RACE预测和验证miR828在番茄中的靶基因。用MegAlign和MEGA5对番茄SlMYB7-like与部分R2R3 MYB转录因子氨基酸序列进行了多重比对和进化树分析。用qRT-PCR分析miR828及其靶基因SlMyb7-like在AC、MicroTom和LA1996 3个不同番茄种质中的表达和miR828在番茄（AC）中的组织特异性表达模式。以miR828过表达转基因番茄和野生番茄为材料，设置正常供磷（+Pi，MS+KH₂PO₄ 3.4 g·L^-1）和缺磷（-Pi，MS+KCl 1.86 g·L^-1）2个处理的培养试验。用qRT-PCR分析miR828及其靶基因在缺磷胁迫下的表达模式。将野生型和miR828过表达的转基因番茄植株缺磷培养15 d后，观察番茄植株地上部分和地下部分的表型差异，通过qRT-PCR分析miR828、miR828的靶基因SlMyb7-like（SGN-U320618）和花青素合成相关酶基因（PAL、CHS、DFR、ANS、3GT和F3′H）的表达模式，应用分光光度计测量各样品的花青素含量。【结果】RLM-5′ RACE剪切试验表明miR828对靶基因SlMyb7-like存在剪切调控作用，且剪切作用位点位于成熟miR828的第10位和第11位核苷酸之间，从而验证了SlMyb7-like是 miR828直接作用的靶基因。氨基酸序列多重比对及进化树分析显示番茄SlMYB7-like与拟南芥AtMYB7和金鱼草MYB330的序列相似性最高。SlMYB7-like含有与花青素合成相关的保守氨基酸基序。miR828在MicroTom幼苗中的表达最高，在LA1996中的表达最低。相反，miR828靶基因SlMyb7-like在LA1996中的表达水平最高，在MicroTom中的表达水平最低。qRT-PCR分析显示SlMyb7-like的表达模式与miR828相反，证明SlMyb7-like被miR828调控。正常供磷条件下，野生型番茄各组织中miR828的表达量普遍较低，但在花芽、花和绿果中相对较高；miR828过表达转基因番茄植株中各花青素合成相关基因的表达量降低为野生型植株的30%—60%，花青素含量降低为野生型植株的40%。在缺磷胁迫下，野生型和miR828过表达转基因番茄植株中miR828及其靶基因SlMyb7-like的表达均受到诱导上调表达；转基因番茄植株地上部分和根系生长受到的抑制程度均小于野生型番茄；转基因番茄植株的颜色较野生型番茄植株颜色浅；转基因番茄植株中SlMyb7-like、花青素合成相关基因的表达以及花青素含量均低于野生型植株；miR828过表达转基因番茄植株缺磷胁迫耐受性却高于野生型番茄。【结论】在番茄中，miR828直接作用的靶基因是SlMyb7-like。野生型番茄所测组织中miR828的表达量普遍较低，但在花芽、花和绿果中相对较高。miR828及其靶基因SlMyb7-like的表达受缺磷胁迫诱导。在正常供磷和缺磷条件下，miR828过表达转基因番茄植株中各花青素合成相关基因的表达量和花青素含量均低于野生型，表明miR828通过靶向SlMyb7-like负调控花青素合成相关基因的表达，进而负调控番茄植株中花青素的生物合成。miR828能够提高番茄对缺磷胁迫的耐受性。

关键词: 番茄, 缺磷胁迫, miR828, SlMyb7-like, 花青素

Abstract: 【Objective】 In this study, the responses of miR828 and its targets to phosphate deficiency and the role of miR828 in anthocyanin biosynthesis under phosphate deficiency were investigated by using wild-type and miR828 overexpressed transgenic tomatoes. 【Method】 The potential targets of miR828 were predicted and validated by psRNATarget and RLM-5′RACE, respectively. An alignment of deduced amino acid sequences of SlMYB7-like with orthologs from Arabidopsis thaliana (AtMYB7) and Antirrhinum majus (AmMYB330) was constructed using MegAlign of DNAStar. The phylogenetic tree of SlMYB7-like and the selected R2R3 MYBs was constructed using neighbor-joining method using MEGA5. The expressions of miR828 and SlMyb7-like in AC, MicroTom and LA1996 tomato seedlings were analyzed. The expression of miR828 in different tissues/organs of tomato (AC) was analyzed by qRT-PCR. Wild-type and miR828 overexpressed transgenic tomatoes were cultured under normal phosphate (KH₂PO₄ 3.4 g·L^-1) and phosphate deficiency (KCl 1.86 g·L^-1) for 15 d. The phenotypic changes and the expression of miR828, SlMyb7-like (SGN-U320618), several anthocyanin biosynthetic genes as well as the anthocyanin content were detected. 【Result】SlMyb7-like was validated to be a direct target of miR828. Protein sequence analysis showed that the SlMYB7-like protein shares the highest homology with the Arabidopsis subgroup 4 MYB7 (AtMYB7), and Snapdragon MYB330, having more than 80% sequence similarity. Phylogenetic analysis grouped SlMYB7-like in a clade with AtMYB7 and AmMYB330. SlMYB7-like contains the conserved amino acid motif ([D/E]L_X2[R/K]_X3L_X6L_X3R/DLIVRLHSLLGNRWSLIAGR), a signature feature common to subgroup 6 R2R3 MYBs (AtMYB75/90/113) that are involved in anthocyanin biosynthesis. The highest abundance of miR828 was detected in MicroTom seedlings, where the transcript of its target gene (SlMyb7-like) was accumulated the lowest. The expression of miR828 in different tissues of tomato tested was very low with relatively higher levels in buds, flowers and green fruits. Under the normal phosphate condition, the expression of anthocyanin biosynthetic genes in the miR828 over-expressed tomatoes decreased by 30%-60% and the content of anthocyanin decreased by 40%. Phosphate deficiency induced both the expression of miR828 and SlMyb7-like. The expression of SlMyb7-like, anthocyanin biosynthetic genes and the content of anthocyanin in the miR828 overexpressed tomatoes were lower than that in control plants under phosphate deficiency. The above data suggest that miR828 negatively regulates the anthocyanin biosynthesis in tomato under phosphate deficiency.【Conclusion】SlMyb7-like is a direct target gene of miR828. Both the expressions of miR828 and SlMyb7-like are induced under phosphate deficiency. MiR828 negatively controls anthocyanin pathway by repressing the expression of SlMyb7-like, and thereby negatively regulating anthocyanin biosynthetic genes in tomato under phosphate deficiency.

Key words: tomato, phosphate deficiency, miR828, SlMyb7-like, anthocyanin

贾小云，刘慧，沈洁，李芳，丁娜，孙岩，高昌勇，李润植. MicroRNA828负调控缺磷胁迫诱导的番茄花青素生物合成[J]. 中国农业科学, 2015, 48(15): 2911-2924.

JIA Xiao-yun, LIU Hui, SHEN Jie, LI Fang, DING Na, SUN Yan, GAO Chang-yong, LI Run-zhi. Negative Regulation of Anthocyanin Biosynthesis in Tomato by MicroRNA828 Under Phosphate Deficiency[J]. Scientia Agricultura Sinica, 2015, 48(15): 2911-2924.

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参考文献

[1] Mes P J, Boches P, Myers J R, Durst R. Characterization of tomatoes expressing anthocyanin in the fruit. Journal of the American Society for Horticultural Science, 2008, 133: 262-269.

[2] Gou J Y, Felippes F F, Liu C J, Weigel D, Wang J W. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. The Plant Cell,2011, 23: 1512-1522.

[3] Jones-Rhoades M W, Bartel D P, Bartel B. MicroRNAs and their regulatory roles in plants. Annual Review of Plant Biology, 2006, 57: 19-53.

[4] Matzke M, Kanno T, Daxinger L, Huettel B, Matzke A J. RNA-mediated chromatin-based silencing in plants. Current Opinion in Cell Biology,2009, 21: 367-376.

[5] Xia R, Zhu H, An Y Q, Beers E P, Liu Z. Apple miRNAs and tasiRNAs with novel regulatory networks. Genome Biology, 2012, 13: R47.

[6] Zuluaga D L, Gonzali S, Loreti E, Pucciariello C, Degl'innocenti E, Guidi L, Alpi A, Perata P. Arabidopsis thaliana MYB75/PAP1 transcription factor induces anthocyanin production in transgenic tomato plants. Functional Plant Biology,2008, 35: 606-618.

[7] Butelli E, Titta L, Giorgio M, Mock H, Matros A, Peterek S, Schijlen E G, Hall R D, Bovy A G, Luo J, Martin C. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology,2008, 26: 1301-1308.

[8] Sunkar R, Zhu J K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. The Plant Cell, 2004, 16(8): 2001-2019.

[9] Bari R, Datt Pant B, Stitt M, Scheible W R. PHO2, microRNA399 and PHR1 define a phosphate-signaling pathway in plants. Plant Physiology, 2006, 141(3): 988-999.

[10] Chiou T J, Aung K, Lin S I, Wu C C, Chiang S F, Su C L. Regulation of phosphate homeostasis by microRNA in Arabidopsis. The Plant Cell, 2006, 18: 412-421.

[11] Rajagopalan R, Vaucheret H, Trejo J, Bartel D P. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes & Development, 2006, 20: 3407-3425.

[12] Moxon S, Jing R, Szittya G, Schwach F, Rusholme Pilcher R L, Moulton V, Dalmay T. Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome research, 2008, 18: 1602-1609.

[13] Guan X, Pang M, Nah G, Shi X, Ye W, Stelly D M, Chen Z J. miR828 and miR858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development. Nature Communications, 2014, 5: 3050.

[14] Luo Q J, Mittal A, Jia F, Rock C D. An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars in Arabidopsis. Plant Molecular Biology, 2012, 80: 117-129.

[15] Hsieh L C, Lin S I, Shih A C, Chen J W, Lin W Y, Tseng C Y, Li W H, Chiou T J. Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing. Plant Physiology, 2009, 151: 2120-2132.

[16] Borevitz J O, Xia Y, Blount J, Dixon R A, Lamb C. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. The Plant Cell,2000, 12: 2383-2394.

[17] Yang F X, Cai J, Yang Y, Liu Z B. Overexpression of microRNA828 reduces anthocyanin accumulation in Arabidopsis. Plant Cell Tissue Organ Culture, 2013, 115: 159-167.

[18] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 2001, 25: 402-408.

[19] 谢烨, 孙毅, 李淡宁, 黄继荣. 拟南芥MicroRNA828负调控蔗糖诱导的花青素合成. 植物生理学报, 2013, 49(2): 188-194.

Xie Y, Sun Y, Li D N, Huang J R. MicroRNA828 negatively regulates sucrose-induced anthocyanin biosynthesis in Arabidopsis. Plant Physiology Journal, 2013, 49(2): 188-194. (in Chinese)

[20] Lin J S, Lin C C, Lin H H, Chen Y C, Jeng S T. MicroR828 regulates lignin and H₂O₂ accumulation in sweet potato on wounding. New Phytologist, 2012, 196: 427-440.

[21] 周芳名. 丹参micoR828全长基因的克隆、表达分析及遗传转化[D]. 重庆: 西南大学, 2013.

Zhou F M. Cloning, expression analysis and genetic transformation of microR828 in Salvia miltiorrhiza[D]. Chongqing: Southwest University, 2013. (in Chinese)

[22] 刘伟伟, 于丽丽, 方媛, 周莹, 李洋, 周波. 番茄microRNA828的克隆表达及其靶基因的鉴定. 园艺学报, 2015, 42(1): 47-55.

Liu W W, Yu L L, Fang Y, Zhou Y, Li Y, Zhou B. Clone and expression analysis of LemiR828 and identification of its target gene in tomato. Acta Horticulturae Sinica, 2015, 42(1): 47-55. (in Chinese)

[23] Chen H M, Chen L T, Patel K, Li Y H, Baulcombe D C, Wu S H. 22-nucleotide RNAs trigger secondary siRNA biogenesis in plants. Proceedings of the National Academy of Sciences of the United States of America,2010, 107: 15269-15274.

[24] Jia X Y, Shen J, Liu H, Li F, Ding N, Gao C Y, Pattanaik P, Patra P, Li R Z, Yuan L.Small tandem target mimic-mediated blockage of microRNA858 induces anthocyanin accumulation in tomato. Planta, 2015, 242(1): 283-293.