18 miRNAs in Larix kaempferi During Seed Germination

WU Tao; HAN Su-ying; ZHANG Jun-hong; LI Wan-feng; YANG Wen-hua; QI Li-wang

Volume 26 Issue S1

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18 miRNAs in Larix kaempferi During Seed Germination

1.
Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
2.
Yunnan Academy of Forestry, Yunnan Key Laboratory for Forest Plant Cultivation and Utilization, Kunming 650201, Yunnan, China
3.
Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China

Received Date: 2013-08-24

Abstract

The seed of Larix kaempferi (Siebold. & Zucc.) Gordon was used as material to investigate the potential molecular regulation mechanisms of miRNA during seed germination. The germination process of L.kaempferi seed includes the following: The imbibition of dry seed was completed after 24 hours of socking; the testa rupture occurred after 5 days and followed by radicle emergence (videlicet, endosperm rupture) after 9 days of germination culture; the cotyledons expanded fully after 28 days. The expression level of 18 miRNAs was relatively quantified using qRT-PCR technology among five key seed germinating stages, which including dry seed (0 day after germinating), imbibed seed (1st day after germinating), testa rupture (5th day), radicle emergence (9th day) and cotyledon extension (28th day). The results are as follows. (1)The expression levels of 18 miRNAs were the highest at dry seed stage and then went down rapidly at imbibed seed and testa rupture stage. The miRNAs were highly expressed in dry seed stage than in subsequent stages. The expression level in dry seed stage was 4.8 to 43.5 times, 17.2 to 1 000 times, 45.5 to 1 000 times and 62.5 to 1 862.2 times that of those in imbibed seed, testa rupture, radicle emergence and extension cotyledon stage, respectively. (2)The expression level of miR894 was the highest and that of miR408 was the lowest in each stage; and the others fluctuated, ranking from high to low as miR951, miR950, miR156, miR159, miR396, miR398, miR390, miR160, miR947, miR165, miR319, miR162, miR171, miR397, miR395 and miR172. (3)The target genes and their possible functions predicted using bioinformatics methods were responsible for the expression of a great number of binding proteins, cellular components and MAP kinase proteins associated with seed germination. (4) JR160237 was the target gene of miR160, and the cleaved fragments mapped exactly to the predicted cleavage site (between nucleotides 10 and 11 from the 5'end of miR160). JR160237 mRNA reached the peak at imbibed seed stage, then decreased and dynamically expressed at following stages with small fluctuation.
- miRNA,
- Larix kaempferi,
- seed,
- germination,
- qRT-PCR

References

[1]	Chuck G S, Tobias C, Sun L, et al. Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass[J]. Proc Natl Acad Sci USA, 2011, 108(42): 17550-17555
[2]	Zhang B, Pan X, Cobb G P, et al. Plant microRNA: a small regulatory molecule with big impact[J]. Dev Biol, 2006, 289(1): 3-16
[3]	Reyes J L, Chua N H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination[J]. Plant J, 2007, 49(4): 592-606
[4]	Liu P P, Montgomery T A, Fahlgren N, et al. Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages[J]. Plant J, 2007, 52(1): 133-146
[5]	Bentsink L, Koornneef M. Seed dormancy and germination[J]. Arabidopsis Book, 2008, 6: e0119
[6]	Holdsworth M J, Bentsink L, Soppe W J. Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination[J]. New Phytol, 2008, 179(1): 33-54
[7]	Penfield S, King J. Towards a systems biology approach to understanding seed dormancy and germination[J]. Proc Biol Sci, 2009, 276(1673): 3561-3569
[8]	Seo M, Jikumaru Y, Kamiya Y. Profiling of hormones and related metabolites in seed dormancy and germination studies[J]. Methods Mol Biol, 2011, 773: 99-111
[9]	马常耕, 孙晓梅. 我国落叶松遗传改良现状及发展方向[J].世界林业研究, 2008, 21(3): 58-63
[10]	常培英, 刘曼玲. 华北落叶松种子发芽促进和发芽指标的研究[J].林业科学, 1989, 25(2): 157-161
[11]	Zhang J H, Zhang S G, Han S Y, et al. Genome-wide identification of microRNAs in Japanese larch and stage-specific modulation of eleven conserved microRNAs and their targets during somatic embryogenesis[J]. Planta, 2012, 236(2): 647-657
[12]	Zhang J H, Wu T, Li L, et al. Dynamic expression of small RNA populations in larch (Larix leptolepis)[J]. Planta, 2013, 237(1): 89-101
[13]	Shi R, Chiang V L. Facile means for quantifying microRNA expression by real-time PCR[J]. Biotechniques, 2005, 39(4): 519-525
[14]	Dai X, Zhao P X. psRNATarget: a plant small RNA target analysis server[J]. Nucleic Acids Res, 2011, 39(Web Server issue): W155-159
[15]	Sunkar R, Girke T, Jain P K, et al. Cloning and characterization of microRNAs from rice[J]. Plant Cell, 2005, 17(5): 1397-1411
[16]	Willmann M R, Mehalick A J, Packer R L, et al. MicroRNAs regulate the timing of embryo maturation in Arabidopsis[J]. Plant Physiol, 2011,155(4):1871-1884
[17]	Wang L, Liu H, Li D, et al. Identification and characterization of maize microRNAs involved in the very early stage of seed germination[J]. BMC Genomics, 2011, 12: 154
[18]	尚杰,王文章,赵垦田. 白皮松种子萌发过程中有机质代谢的研究[J].东北林业大学学报, 1992, 20(2): 24-29
[19]	王国华, 时培玲, 石长德. 樟子松种子萌发过程中碳水化合物代谢变化规律[J].防护林科技, 2011(2): 32-33
[20]	李志芬, 王国华, 杨扬. 樟子松种子萌发过程中脂肪和蛋白质代谢的变化规律[J]. 防护林科技, 2011(1): 53-55
[21]	Mallory A C, Reinhart B J, Bartel D, et al. A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco[J]. Proc Natl Acad Sci USA, 2002, 99(23): 15228-15233
[22]	Kasschau K D, Xie Z, Allen E, et al. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function[J]. Dev Cell, 2003, 4(2): 205-217
[23]	Trindade I, Capitao C, Dalmay T, et al. miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula[J]. Planta, 2010, 231(3): 705-716
[24]	Pantaleo V, Szittya G, Moxon S, et al. Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis[J]. Plant J, 2010, 62(6): 960-976
[25]	Kantar M, Lucas S J, Budak H. miRNA expression patterns of Triticum dicoccoides in response to shock drought stress[J]. Planta, 2011, 233(3): 471-484

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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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18 miRNAs in Larix kaempferi During Seed Germination

1. Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

2. Yunnan Academy of Forestry, Yunnan Key Laboratory for Forest Plant Cultivation and Utilization, Kunming 650201, Yunnan, China

3. Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China

Received Date: 2013-08-24

Abstract: The seed of Larix kaempferi (Siebold. & Zucc.) Gordon was used as material to investigate the potential molecular regulation mechanisms of miRNA during seed germination. The germination process of L.kaempferi seed includes the following: The imbibition of dry seed was completed after 24 hours of socking; the testa rupture occurred after 5 days and followed by radicle emergence (videlicet, endosperm rupture) after 9 days of germination culture; the cotyledons expanded fully after 28 days. The expression level of 18 miRNAs was relatively quantified using qRT-PCR technology among five key seed germinating stages, which including dry seed (0 day after germinating), imbibed seed (1st day after germinating), testa rupture (5th day), radicle emergence (9th day) and cotyledon extension (28th day). The results are as follows. (1)The expression levels of 18 miRNAs were the highest at dry seed stage and then went down rapidly at imbibed seed and testa rupture stage. The miRNAs were highly expressed in dry seed stage than in subsequent stages. The expression level in dry seed stage was 4.8 to 43.5 times, 17.2 to 1 000 times, 45.5 to 1 000 times and 62.5 to 1 862.2 times that of those in imbibed seed, testa rupture, radicle emergence and extension cotyledon stage, respectively. (2)The expression level of miR894 was the highest and that of miR408 was the lowest in each stage; and the others fluctuated, ranking from high to low as miR951, miR950, miR156, miR159, miR396, miR398, miR390, miR160, miR947, miR165, miR319, miR162, miR171, miR397, miR395 and miR172. (3)The target genes and their possible functions predicted using bioinformatics methods were responsible for the expression of a great number of binding proteins, cellular components and MAP kinase proteins associated with seed germination. (4) JR160237 was the target gene of miR160, and the cleaved fragments mapped exactly to the predicted cleavage site (between nucleotides 10 and 11 from the 5'end of miR160). JR160237 mRNA reached the peak at imbibed seed stage, then decreased and dynamically expressed at following stages with small fluctuation.

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Reference (25)

[1]	Chuck G S, Tobias C, Sun L, et al. Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass[J]. Proc Natl Acad Sci USA, 2011, 108(42): 17550-17555
[2]	Zhang B, Pan X, Cobb G P, et al. Plant microRNA: a small regulatory molecule with big impact[J]. Dev Biol, 2006, 289(1): 3-16
[3]	Reyes J L, Chua N H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination[J]. Plant J, 2007, 49(4): 592-606
[4]	Liu P P, Montgomery T A, Fahlgren N, et al. Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages[J]. Plant J, 2007, 52(1): 133-146
[5]	Bentsink L, Koornneef M. Seed dormancy and germination[J]. Arabidopsis Book, 2008, 6: e0119
[6]	Holdsworth M J, Bentsink L, Soppe W J. Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination[J]. New Phytol, 2008, 179(1): 33-54
[7]	Penfield S, King J. Towards a systems biology approach to understanding seed dormancy and germination[J]. Proc Biol Sci, 2009, 276(1673): 3561-3569
[8]	Seo M, Jikumaru Y, Kamiya Y. Profiling of hormones and related metabolites in seed dormancy and germination studies[J]. Methods Mol Biol, 2011, 773: 99-111
[9]	马常耕, 孙晓梅. 我国落叶松遗传改良现状及发展方向[J].世界林业研究, 2008, 21(3): 58-63
[10]	常培英, 刘曼玲. 华北落叶松种子发芽促进和发芽指标的研究[J].林业科学, 1989, 25(2): 157-161
[11]	Zhang J H, Zhang S G, Han S Y, et al. Genome-wide identification of microRNAs in Japanese larch and stage-specific modulation of eleven conserved microRNAs and their targets during somatic embryogenesis[J]. Planta, 2012, 236(2): 647-657
[12]	Zhang J H, Wu T, Li L, et al. Dynamic expression of small RNA populations in larch (Larix leptolepis)[J]. Planta, 2013, 237(1): 89-101
[13]	Shi R, Chiang V L. Facile means for quantifying microRNA expression by real-time PCR[J]. Biotechniques, 2005, 39(4): 519-525
[14]	Dai X, Zhao P X. psRNATarget: a plant small RNA target analysis server[J]. Nucleic Acids Res, 2011, 39(Web Server issue): W155-159
[15]	Sunkar R, Girke T, Jain P K, et al. Cloning and characterization of microRNAs from rice[J]. Plant Cell, 2005, 17(5): 1397-1411
[16]	Willmann M R, Mehalick A J, Packer R L, et al. MicroRNAs regulate the timing of embryo maturation in Arabidopsis[J]. Plant Physiol, 2011,155(4):1871-1884
[17]	Wang L, Liu H, Li D, et al. Identification and characterization of maize microRNAs involved in the very early stage of seed germination[J]. BMC Genomics, 2011, 12: 154
[18]	尚杰,王文章,赵垦田. 白皮松种子萌发过程中有机质代谢的研究[J].东北林业大学学报, 1992, 20(2): 24-29
[19]	王国华, 时培玲, 石长德. 樟子松种子萌发过程中碳水化合物代谢变化规律[J].防护林科技, 2011(2): 32-33
[20]	李志芬, 王国华, 杨扬. 樟子松种子萌发过程中脂肪和蛋白质代谢的变化规律[J]. 防护林科技, 2011(1): 53-55
[21]	Mallory A C, Reinhart B J, Bartel D, et al. A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco[J]. Proc Natl Acad Sci USA, 2002, 99(23): 15228-15233
[22]	Kasschau K D, Xie Z, Allen E, et al. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function[J]. Dev Cell, 2003, 4(2): 205-217
[23]	Trindade I, Capitao C, Dalmay T, et al. miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula[J]. Planta, 2010, 231(3): 705-716
[24]	Pantaleo V, Szittya G, Moxon S, et al. Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis[J]. Plant J, 2010, 62(6): 960-976
[25]	Kantar M, Lucas S J, Budak H. miRNA expression patterns of Triticum dicoccoides in response to shock drought stress[J]. Planta, 2011, 233(3): 471-484

18 miRNAs in Larix kaempferi During Seed Germination

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通讯作者: 陈斌, bchen63@163.com

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18 miRNAs in Larix kaempferi During Seed Germination

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