Expression Analysis of miR164b and Its Target Gene PeNAC1 in Phyllostachys edulis under Stress

WANG Li-li; ZHAO Han-sheng; SUN Hua-yu; DONG Li-li; LOU Yong-feng; GAO Zhi-min

Volume 28 Issue 5

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Expression Analysis of miR164b and Its Target Gene PeNAC1 in Phyllostachys edulis under Stress

1.
International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102, China

Received Date: 2014-07-31

Abstract

MicroRNAs (miRNAs) play important roles in a variety of biological growth processes and stress-resistance responses. As the plant-specific miRNA family, the miR164 mainly targets for NAC transcriptional factors. To reveal the regulatory mechanism for miR164 and its target gene, the mature sequence of miR164b and its targeted sequence of PeNAC1 were isolated respectively from Phyllostachys edulis seedlings by RT-PCR with stem-loop RT primers. The analysis indicated that there was one miR164b complementary site located in the open reading frame region of PeNAC1 mRNA. The sequencing result of RLM-5'RACE confirmed that PeNAC1 was regulated by miR164b through specific cleavage at the site between the 10th and 11th bases. Tissue specific expression of miR164b and PeNAC1 demonstrated that they both expressed in root, stem, leaf and sheath, of which miR164b expressed in root with the highest level and the lowest level in stem, while that of PeNAC1 coincided with miR164b conversely. Real-time quantitative PCR analysis showed that miR164b was down-regulated under the treatments of NaCl (250 mmol·L-1), low temperature (4℃) and high light (1 500 μmol·m-2·s-1), but it was up-regulated obviously under the treatment of GA3 (100 μmol·L-1). Meanwhile, the expression of PeNAC1 showed exactly the opposite trends. It is suggested that miR164b played a regulator role in the expression of PeNAC1, which might be closely related to the resilience process of respond to abiotic stress. This result provides a reference of bamboo molecular breeding for resilience by using miRNA.
- Phyllostachys edulis,
- miRNA,
- NAC transcriptional factor,
- abiotic stress

References

[1]	Llave C, Xie Z, Kasschau K DM, et al. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA[J]. Science, 2002, 297(5589):2053-2056.
[2]	Sunkar R. MicroRNAs with macro-effects on plant stress responses[J]. Seminars in Cell and Developmental Biology, 2010, 21:805-811.
[3]	Sunkar R, Zhu J K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis[J]. Plant Cell, 2004, 16(8):2001-2019.
[4]	Zhao B T, Liang R Q, Ge L F, et al. Identification of drought-induced microRNAs in rice[J]. Biochemical Biophysical Research Communications, 2007, 354(2):585-590.
[5]	Lu S, Sun Y H, Chiang V L. Stress-responsive microRNAs in Populus[J]. Plant Journal, 2008, 55(1):131-151.
[6]	Gao P, Bai X, Yang L, Lv D, et al. Osa-MIR393:A salinity- and alkaline stress-related microRNA gene[J]. Molecular Biology Reports, 2011, 38(1):237-242.
[7]	Zhang X, Zou Z, Gong P, et al. Over-expression of microRNA169 confers enhanced drought tolerance to tomato[J]. Biotechnology Letters, 2011, 33(2):403-409.
[8]	Zhao H S, Chen D L, Peng Z H, et al. Identification and characterization of microRNAs in the leaf of ma bamboo (Dendrocalamus latiflorus) by deep sequencing[J]. PLoS One, 2013, (10):e78755.
[9]	Zhao H, Wang L, Dong L, et al. Discovery and comparative profiling of microRNAs in representative monopodial bamboo (Phyllostachys edulis) and sympodial bamboo (Dendrocalamus latiflorus)[J]. PLoS One, 2014, 9(7):e102375.
[10]	Xu P, Mohorianu I, Yang L, et al. Small RNA profile in moso bamboo root and leaf obtained by high definition adapters[J]. PLoS ONE, 2014, 9(7):e103590.
[11]	Olsen A N, Ernst H A, Leggio L L, et al. NAC transcription factors:structurally distinct, functionally diverse[J]. Trends Plant Science, 2005, 10(2):79-87.
[12]	孙利军, 李大勇, 张慧娟. NAC 转录因子在植物抗病和抗非生物胁迫反应中的作用[J]. 遗传, 2012, 34(8):993-1002.
[13]	高志民, 范少辉, 高健, 等. 基于CTAB法提取毛竹基因组DNA的探讨[J]. 林业科学研究, 2006, 19(6):725-728.
[14]	Gao Z M, Li X P, Li L B, et al. An effective method for total RNA isolation from bamboo[J].Chinese Forest Science Technology, 2006, 5(3):52-54.
[15]	Chen C, Ridzon D A, Gurgler K J, et al. Real-time quantification of microRNAs by stem-loop RT-PCR[J]. Nucleic Acids Research, 2005, 33(20):e179.
[16]	王京京, 童再康, 黄程前, 等.巨桉 EgrCBF1和EgrCBF2 基因的克隆和胁迫响应表达分析[J]. 林业科学, 2012, 48(10):42-48.
[17]	Kayal E W, Navarro M, Marque G, et al. Expression profile of CBF-like transcriptional factor genes from Eucalyptus in response to cold[J]. Journal of Experimental Botany, 2006, 57(10):2455-2496.
[18]	Ding Y, Chen Z, Zhu C. Microarray-based analysis of cadmium-responsive microRNAs in rice (Oryza sativa)[J]. Journal of Experimental Botany, 2011, 62(10):3563-3573.
[19]	Fan C, Ma J, Guo Q, et al. Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis)[J]. PLoS One, 2013, 8(2):e56573.
[20]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitati瑶?挠敐汃汒?摡敮慤琠桴?楥渠瘲漼汳癵楰渾札?涳楃剴????楰渾??牥慴扨楯摤潛灊獝椮猠孍?嵴??卤捳椬攠渲挰攰??㈠??????名?????????ひ????そ????戬爠?嬠??崠??町漬???匠??塎楁敃?兕???旍楻??????敛瑊?愮氠???榯掃爟潩剦亥?ㄠ???搱椬爠攱挷琨猴?为????洶到丶??换汲放慛瘲愲杝攠?瑩漠?搬漠睇湵牯攠杇甬氠慇瑵敯?慗甬砠楥湴?獡楬朮渠慍汩獒?晁漱父?氭慤瑩敲牥慣汴?牤漠潣瑬?摡敶癡敧汥漠灯浦攠湚瑭孎?嵃??偣汯慮湦瑥??攠汬污???ちぬ???????????????扮牴?孩??嵭?卩楺敥戠攨牚?偡??坡敹汳氠浌攮爩?????桂敍祃猠敐汬楡湮捴欠????敯瑧?愬氠?‰刱攲搬甠渲搱愨渱挲礩?愲渲搰?猼灢敲挾楛愲氳楝稠慁瑣楨潡湲?愠浐漬渠杈?灲汲愠湁琬?浂楡捵牬潣副乭?獥?牄潃氬攠?潴映?瑬栮攠???創????景慮洠楯汦礠?楬湯?摡敬瘠敤汥潶灥浬敯湰瑭慥汮?爠潢批甠獡琠湧敩獢獢孥?嵥???敮瘭敲汥潧灵浬敡湴瑥???どっ???????????づ????ば?づ?t, 2004, 131(14):3357-3365.
[21]	Reyes J L, Chua N H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination[J]. Plant Journal, 2007, 49(4):592-606.
[22]	Liu Q, Zhang Y C, Wang C Y, et al. Expression analysis of phytohormone-regulated microRNAs in rice, implying their regulation roles in plant hormone signaling[J]. FEBS Letters, 2009, 583(4):723-728.
[23]	Fujita M, Fujita Y, Maruyama K, et al. A dehydration induced NAC protein, RD26, is involved in a novel ABA dependent stress signaling pathway[J]. Plant Journal, 2004, 39(6):863-876.
[24]	Khraiwesh B, Zhu J K, Zhu J. Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants[J]. Biochimica et Biophysica Acta, 2012, 1819(2):137-148.
[25]	Meng Y, Shao C, Ma X, et al. Expression-based functional investigation of the organ-specific microRNAs in Arabidopsis[J]. PLoS One, 2012, 7(11):e50870.
[26]	Han R, Jian C, Lv J, et al. Identification and characterization of microRNAs in the flag leaf and developing seed of wheat (Triticum aestivum L.)[J]. BMC Genomics, 2014, 15:289.
[27]	Yao Y Y, Guo G G, Ni J F, et al. Cloning and characterization of microRNAs from wheat (Triticum aestivum L.)[J]. Genome Biology, 2007, 8(6):R96.
[28]	谢卡斌. 水稻全长cDNA 文库的构建和两个microRNA的功能研究[J].华中农业大学, 2008.
[29]	Nikovics K, Blein T, Peaucelle A, et al. The balance between the MIR164a and CUC2 genes controls leaf margin serration in Arabidopsis[J]. Plant Cell, 2006, 18(11):2929-2945.
[30]	Kim J H, Woo H R, Kim J, et al. Trifurcate feed forward regulation of age-dependen

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Expression Analysis of miR164b and Its Target Gene PeNAC1 in Phyllostachys edulis under Stress

1. International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102, China

Received Date: 2014-07-31

Abstract: MicroRNAs (miRNAs) play important roles in a variety of biological growth processes and stress-resistance responses. As the plant-specific miRNA family, the miR164 mainly targets for NAC transcriptional factors. To reveal the regulatory mechanism for miR164 and its target gene, the mature sequence of miR164b and its targeted sequence of PeNAC1 were isolated respectively from Phyllostachys edulis seedlings by RT-PCR with stem-loop RT primers. The analysis indicated that there was one miR164b complementary site located in the open reading frame region of PeNAC1 mRNA. The sequencing result of RLM-5'RACE confirmed that PeNAC1 was regulated by miR164b through specific cleavage at the site between the 10th and 11th bases. Tissue specific expression of miR164b and PeNAC1 demonstrated that they both expressed in root, stem, leaf and sheath, of which miR164b expressed in root with the highest level and the lowest level in stem, while that of PeNAC1 coincided with miR164b conversely. Real-time quantitative PCR analysis showed that miR164b was down-regulated under the treatments of NaCl (250 mmol·L-1), low temperature (4℃) and high light (1 500 μmol·m-2·s-1), but it was up-regulated obviously under the treatment of GA3 (100 μmol·L-1). Meanwhile, the expression of PeNAC1 showed exactly the opposite trends. It is suggested that miR164b played a regulator role in the expression of PeNAC1, which might be closely related to the resilience process of respond to abiotic stress. This result provides a reference of bamboo molecular breeding for resilience by using miRNA.

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Reference (30)

[1]	Llave C, Xie Z, Kasschau K DM, et al. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA[J]. Science, 2002, 297(5589):2053-2056.
[2]	Sunkar R. MicroRNAs with macro-effects on plant stress responses[J]. Seminars in Cell and Developmental Biology, 2010, 21:805-811.
[3]	Sunkar R, Zhu J K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis[J]. Plant Cell, 2004, 16(8):2001-2019.
[4]	Zhao B T, Liang R Q, Ge L F, et al. Identification of drought-induced microRNAs in rice[J]. Biochemical Biophysical Research Communications, 2007, 354(2):585-590.
[5]	Lu S, Sun Y H, Chiang V L. Stress-responsive microRNAs in Populus[J]. Plant Journal, 2008, 55(1):131-151.
[6]	Gao P, Bai X, Yang L, Lv D, et al. Osa-MIR393:A salinity- and alkaline stress-related microRNA gene[J]. Molecular Biology Reports, 2011, 38(1):237-242.
[7]	Zhang X, Zou Z, Gong P, et al. Over-expression of microRNA169 confers enhanced drought tolerance to tomato[J]. Biotechnology Letters, 2011, 33(2):403-409.
[8]	Zhao H S, Chen D L, Peng Z H, et al. Identification and characterization of microRNAs in the leaf of ma bamboo (Dendrocalamus latiflorus) by deep sequencing[J]. PLoS One, 2013, (10):e78755.
[9]	Zhao H, Wang L, Dong L, et al. Discovery and comparative profiling of microRNAs in representative monopodial bamboo (Phyllostachys edulis) and sympodial bamboo (Dendrocalamus latiflorus)[J]. PLoS One, 2014, 9(7):e102375.
[10]	Xu P, Mohorianu I, Yang L, et al. Small RNA profile in moso bamboo root and leaf obtained by high definition adapters[J]. PLoS ONE, 2014, 9(7):e103590.
[11]	Olsen A N, Ernst H A, Leggio L L, et al. NAC transcription factors:structurally distinct, functionally diverse[J]. Trends Plant Science, 2005, 10(2):79-87.
[12]	孙利军, 李大勇, 张慧娟. NAC 转录因子在植物抗病和抗非生物胁迫反应中的作用[J]. 遗传, 2012, 34(8):993-1002.
[13]	高志民, 范少辉, 高健, 等. 基于CTAB法提取毛竹基因组DNA的探讨[J]. 林业科学研究, 2006, 19(6):725-728.
[14]	Gao Z M, Li X P, Li L B, et al. An effective method for total RNA isolation from bamboo[J].Chinese Forest Science Technology, 2006, 5(3):52-54.
[15]	Chen C, Ridzon D A, Gurgler K J, et al. Real-time quantification of microRNAs by stem-loop RT-PCR[J]. Nucleic Acids Research, 2005, 33(20):e179.
[16]	王京京, 童再康, 黄程前, 等.巨桉 EgrCBF1和EgrCBF2 基因的克隆和胁迫响应表达分析[J]. 林业科学, 2012, 48(10):42-48.
[17]	Kayal E W, Navarro M, Marque G, et al. Expression profile of CBF-like transcriptional factor genes from Eucalyptus in response to cold[J]. Journal of Experimental Botany, 2006, 57(10):2455-2496.
[18]	Ding Y, Chen Z, Zhu C. Microarray-based analysis of cadmium-responsive microRNAs in rice (Oryza sativa)[J]. Journal of Experimental Botany, 2011, 62(10):3563-3573.
[19]	Fan C, Ma J, Guo Q, et al. Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis)[J]. PLoS One, 2013, 8(2):e56573.
[20]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitati瑶?挠敐汃汒?摡敮慤琠桴?楥渠瘲漼汳癵楰渾札?涳楃剴????楰渾??牥慴扨楯摤潛灊獝椮猠孍?嵴??卤捳椬攠渲挰攰??㈠??????名?????????ひ????そ????戬爠?嬠??崠??町漬???匠??塎楁敃?兕???旍楻??????敛瑊?愮氠???榯掃爟潩剦亥?ㄠ???搱椬爠攱挷琨猴?为????洶到丶??换汲放慛瘲愲杝攠?瑩漠?搬漠睇湵牯攠杇甬氠慇瑵敯?慗甬砠楥湴?獡楬朮渠慍汩獒?晁漱父?氭慤瑩敲牥慣汴?牤漠潣瑬?摡敶癡敧汥漠灯浦攠湚瑭孎?嵃??偣汯慮湦瑥??攠汬污???ちぬ???????????????扮牴?孩??嵭?卩楺敥戠攨牚?偡??坡敹汳氠浌攮爩?????桂敍祃猠敐汬楡湮捴欠????敯瑧?愬氠?‰刱攲搬甠渲搱愨渱挲礩?愲渲搰?猼灢敲挾楛愲氳楝稠慁瑣楨潡湲?愠浐漬渠杈?灲汲愠湁琬?浂楡捵牬潣副乭?獥?牄潃氬攠?潴映?瑬栮攠???創????景慮洠楯汦礠?楬湯?摡敬瘠敤汥潶灥浬敯湰瑭慥汮?爠潢批甠獡琠湧敩獢獢孥?嵥???敮瘭敲汥潧灵浬敡湴瑥???どっ???????????づ????ば?づ?t, 2004, 131(14):3357-3365.
[21]	Reyes J L, Chua N H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination[J]. Plant Journal, 2007, 49(4):592-606.
[22]	Liu Q, Zhang Y C, Wang C Y, et al. Expression analysis of phytohormone-regulated microRNAs in rice, implying their regulation roles in plant hormone signaling[J]. FEBS Letters, 2009, 583(4):723-728.
[23]	Fujita M, Fujita Y, Maruyama K, et al. A dehydration induced NAC protein, RD26, is involved in a novel ABA dependent stress signaling pathway[J]. Plant Journal, 2004, 39(6):863-876.
[24]	Khraiwesh B, Zhu J K, Zhu J. Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants[J]. Biochimica et Biophysica Acta, 2012, 1819(2):137-148.
[25]	Meng Y, Shao C, Ma X, et al. Expression-based functional investigation of the organ-specific microRNAs in Arabidopsis[J]. PLoS One, 2012, 7(11):e50870.
[26]	Han R, Jian C, Lv J, et al. Identification and characterization of microRNAs in the flag leaf and developing seed of wheat (Triticum aestivum L.)[J]. BMC Genomics, 2014, 15:289.
[27]	Yao Y Y, Guo G G, Ni J F, et al. Cloning and characterization of microRNAs from wheat (Triticum aestivum L.)[J]. Genome Biology, 2007, 8(6):R96.
[28]	谢卡斌. 水稻全长cDNA 文库的构建和两个microRNA的功能研究[J].华中农业大学, 2008.
[29]	Nikovics K, Blein T, Peaucelle A, et al. The balance between the MIR164a and CUC2 genes controls leaf margin serration in Arabidopsis[J]. Plant Cell, 2006, 18(11):2929-2945.
[30]	Kim J H, Woo H R, Kim J, et al. Trifurcate feed forward regulation of age-dependen

Expression Analysis of miR164b and Its Target Gene PeNAC1 in Phyllostachys edulis under Stress

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通讯作者: 陈斌, bchen63@163.com

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Expression Analysis of miR164b and Its Target Gene PeNAC1 in Phyllostachys edulis under Stress

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