摘要
MicroRNA(miRNA)是一类调节基因转录后表达中起重要作用的非编码RNA。普通野生稻(Oryza rufipogon Griff.)是栽培稻的近缘祖先,是改良栽培稻的重要种质资源。为了揭示普通野生稻向栽培稻驯化过程中miRNA的变化,并克隆普通野生稻中的miRNA,本研究利用Illumina测序技术对海南万宁普通野生稻小RNA文库进行了分离、鉴定及表达分析,对本项研究中发现的只在普通野生稻中出现的miRNA构建植物表达载体,转化水稻品种日本晴,为普通野生稻中miRNA的功能分析打下基础。主要研究结果如下:
1.小RNA文库高通量测序数据分析
利用Illumina高通量测序平台,分析了普通野生稻小RNA文库,共得到高质量序列8558370个,共2307484种;通过对所有的sRNA序列Blastn比对miRNA数据库(miRBase15.0)中已知的miRNA,在普通野生稻中发现了属于25个家族的104种保守的miRNAs,对比了它们在栽培稻中表达情况的异同;对普通野生稻测到次数最多的前四位:miR156,miR168,miR528,miR166,不同于水稻高通量测序的保守miRNA家族的前四位:miR169, miR156, miR168, miR172;发现215个水稻中特异的miRNA,属于78个家族;经UNAFOLD分析预测,353个小RNA对应的水稻基因组序列能形成发卡环二级结构,进一步通过是否有互补链miRNA*存在及二级结构自由能分析,得到了23个新的miRNA;对23个新的miRNA进行靶基因预测,共发现11个新的miRNA有17个靶基因。
2.候选新miRNA的实验鉴定
提取普通野生稻小分子量RNA,利用改进的茎环引物反转录PCR进一步对能形成发卡环二级结构的353个预测的小RNA序列进行了表达分析,结果发现除了确定的有互补链miRNA*存在23个新的miRNA,还有32个序列通过茎环引物反转录之后PCR扩增,连接到克隆载体,测序得到了目的序列片段,说明这32个序列在普通野生稻中有表达,可能也是新的miRNA。对这32个可能的新miRNA进行靶基因预测,23个预测到了靶基因序列。
3.新miRNA的表达分析及转化栽培稻日本晴
利用RT-PCR检测了CWR-miR28,CWR-miR61,CWR-miR64,CWR-miR68,CWR-miR153,CWR-miR200,CWR-miR264,CWR-miR308在普通野生稻中的表达情况。所有的检测对象在所有部位都有表达,CWR-miR28,CWR-miR153,CWR-miR264在根,茎,叶中表达量相当。其它的在不同部位表达有差异。将23个新的miRNA构建过表达载体,通过农杆菌介导法转化栽培稻日本晴,得到了抗性愈伤,为进一步功能分析打下了基础。
MicroRNA(miRNA) are a class of non-coding RNAs involved in post-transcriptional control of gene expression, either via degradation or translational inhibition of target mRNAs. Common wild rice (Oryza rufipogon Griff.) is considered to be the ancestor of Asian cultivated rice species, Oryza sativa L. And it is also a important germplasm for cultivated rice modification. In order to reveal the variation of miRNA during the domestication of cultivated rice and, hopefully, clone common wild rice specific miRNA, we use Illumina sequencing approach to characterize, identify and do expression analysis of sRNA cDNA library. Over expression vector harboring precursor of miRNA which were found to be specific to common wild rice were constructed and transferred into cultivated rice variety Nipponbare via Agrobaterium-nediated method, setting the stage for studying the function of those miRNA. The main results were as follows:
Dada analysis of small RNA library for Illumina sequencing
Using Illumina sequencing technology, we analyze the common wild rice sRNA cDNA library and obtain High quality reads 8558370 consisting of 2307484 unique sequences; Through Aligning small RNA to the miRNA precursor of Oryza sativa in miRBase14.0 we identify 104 miRNA belonging to 25 conserved miRNA families; According to the miRNA count in our study and previously reports the most highly expressed conserved miRNA in common wild rice ranked from high to low is miR156,miR168,miR528,miR166,miR167, while in cultivated rice is miR169, miR156, miR168, miR 172;Besides the conserved miRNAs, we also found 215 Rice specific miRNA belonging to 78 families; Using UNAFOLD we found 353 sRNA sequences seemed to be able to generate fold-back secondary structure, further analysis including the existence of miRNA* and the free energy of the harpin, we identified 23 novel miRNA; 17 target genes of 11 new miRNA were predicted by way of bioinformatics approach.
Validation of new miRNA
After extracted small RNA, we use Stem-loop RT-PCR for detecting the expression of 353 small RNA sequences which were predicted to be able to generate fold-back secondary structure. Among those, by sequencing, we got 32 75bp PCR product containing 21nt small RNA except for 23 new miRNA which have miRNA*. Suggesting those 32 sequences are expressed so they are candidate new miRNA. Using on line software--miRU: Plant miRNA Potential Target Find, 23 out of 32 candidate have predicted targets.
Expression patterns of newly identified miRNA and vector construction and transformation of cultivated rice
Knowledge about the expression of miRNA might provide clues about their functions. We did Semi-quantitative PCR for CWR-miR28 , CWR-miR61 , CWR-miR64 , CWR-miR68 ,CWR-miR153,CWR-miR200,CWR-miR264,CWR-miR308 and found all of them expressed in common wild rice, and all of them show different expression level in root, stem and leaf except for CWR-miR28,CWR-miR153,CWR-miR264,.
In order for further study of functions of new miRNAs, we constructed 23 over expression vector harboring precursor of new miRNAs and transferred them into cultivated rice variety Nipponbare via Agrobaterium-mediated method. Now 2 of them are in the differentiation stage.
引文
1. Achard P., Herr A., Baulcombe, D. C., Harberd N.P. 2004. Modulation of floral development by agibberellin-regulated microRNA. Development 131, 3357–3365.
2. Adai A., Johnson C., Mlotshwa S., Archer-Evans S., Manocha V., Vance V., Sundaresan V. 2005. Computational prediction of miRNAs in Arabidopsis thaliana. Genome Res. 15, 78– 91.
3. Allen E., Xie Z., Gustafson, A.M., Carrington, J.C., 2005. microRNAdirected phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207– 221.
4. Allen E., Xie Z., Gustafson, A.M., Sung, G.H., Spatafora, J.W., and Carrington,J.C. 2004. Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat. Genet. 36, 1282–1290.
5. Aukerman, M.J., Sakai, H.2003. Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15, 2730– 2741
6. Axtell, M.J., and Bowman, J.L. 2008. Evolution of plant microRNAs and their targets. Trends Plant Sci. 13, 343–349.
7. Bao N., Lye K.W., Barton M.K. 2004. MicroRNA binding sites in Arabidopsis class IIIHD-ZIP mRNAs are required for methylation of the template chromosome. Dev. Cell 7, 653– 662.
8. Barbisin M, Xu N L, Mahuyakar V R, Andersen M R, Lao K Q, Livak Bartel, B. 2005. MicroRNAs directing siRNA biogenesis. Nat. Struct. Mol. Biol. 12, 569– 571.
9. Bartel, D.P. 2009. MicroRNAs: target recognition and regulatory functions. Cell., 136, 215-233.
10. Bartel D.P.2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.
11. Bartel D.P., Chen, C.Z. 2004.Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat. Rev. Genet.5, 396-400.
12. Bollman K.M., Aukerman, M.J., Park, M.Y., Hunter, C., Berardini, T.Z., Poethig, R.S.2003. HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development 130, 1493–1504.
13. Bowman J.L. 2004. Class III HD-Zip gene regulation, the golden fleece of ARGONAUTE activity? Bioessays 26, 938–942.
14. Brodersen P., Sakvarelidze-Achard, L.; Bruun-Rasmussen, M., Dunoyer, P.;Yamamoto, Y.Y., Sieburth, L.; Voinnet, O. 2008.Widespread translational inhibition by plant miRNAs and siRNAs. Science, 320, 1185-1190.
15. Brodersen P., Voinnet O. Revisiting the principles of microRNA target recognition and mode of action. 2009. Nat. Rev. Mol. Cell Biol. 10, 141-148.
16. Brown J.R., Sanseau P. 2005. A computational view of microRNAs and their targets. Drug Discov. Today 10, 595– 601.
17. Cameron Johnson, Anna Kasprzewska, Kristin Tennessen, et al. 2009. Clusters and superclustersof phased small RNAs in the developing inflorescence of rice. 7,1-12
18. Cardon G.H., Hohmann S., Nettesheim K., Saedle H., Huijser P. 1997. Functional analysis of the Arabidopsis thaliana SBP-box gene SPL3: a novel gene involved in the floral transition. Plant J. 12, 367–377.
19. Carrington J.C., Ambros V. 2003. Role of microRNAs in plant and animal development. Science 301, 336–338.
20. Caudy A.A., Ketting R.F., Hammond S.M., Denli A.M., Bathoorn A.M., Tops B.B., Silva J.M., Myers M.M., Hannon G.J., Plasterk R.H. 2003. A micrococcal nuclease homologue in RNAi effector complexes. Nature 425, 411– 414.
21. Chapman E.J., Prokhnevsky A.I., Gopinath K., Dolja V.V., Carrington J.C. 2004. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev. 18, 1179– 1186.
22. Chen C F, Ridzon D A, Broomer A J, Zhou Z H, Lee D H, Nguyen J T,Chen, X. A Chen C F, Ridzon D A, Broomer A J, Zhou Z H, Lee D H, Nguyen J T, Barbisin M, Xu N L, Mahuyakar V R, Andersen M R, Lao K Q, Livak K J, Guegler K J. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Research. 2005. 33(20): 179-187.
23. microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. 2004. Science 303, 2022-2025.
24. Chen X. 2004. A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303, 2022–2025.
25. Croce, C.M., Calin, G.A., 2005. miRNAs, cancer, and stem cell division. Cell 122, 6– 7.
26. de Felippes, F.F., Schneeberger K., Dezulian T., Huson D.H., and Weigel D. 2008. Evolution of Arabidopsis thaliana microRNAs from random sequences. RNA 14, 2455–2459
27. de Meaux, J., Hu J.Y., Tartler U., and Goebel U. 2008. Structurally different alleles of the ath-MIR824 microRNA precursor are maintained at high frequency in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 105, 8994–8999.
28. Ding, S.W., 2000. RNA silencing. Curr. Opin. Biotechnol. 11, 152– 156.
29. Dong YB, Kong H, Peng YF. 2008. Flowering and reproduction habits of Oryza rufipogon in Wanning city of Hainan province. J Plant Genet Resour 9:218–222
30. Dugas D.V., Bartel B. 2004. MicroRNA regulation of gene expression in plants. Curr. Opin. Plant Biol. 7, 512–520.
31. Dunoyer, P., Himber, C., Ruiz-Ferrer, V., Alioua, A., and Voinnet, O. 2007. Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways. Nat. Genet. 39, 848–856.
32. Dunoyer, P., Lecellier, C.H., Parizotto, E.A., Himber, C., and Voinnet, O. 2004. Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16, 1235–1250.
33. Emery J.F., Floyd S.K., Alvarez J., Eshed Y., Hawker N.P., Izhaki A., Baum S.F., Bowman J.L.2003. Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes. Curr. Biol. 13, 1768– 1774.
34. Fahlgren N., Howell M.D., Kasschau K.D., Chapman E.J., Sullivan C.M.,Cumbie J.S., Givan, S.A., Law T.F., Grant S.R., Dang J. L., and Carrington,J.C. 2007. High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2, e219. 10.1371/journal.pone.0000219.
35. Floyd S.K., Bowman J.L. 2004. Ancient microRNA target sequences in plants. Nature 428, 485– 486.
36. Garcia D. 2008. A miRacle in plant development: Role of microRNAs in cell differentiation and patterning. Semin. Cell Dev. Biol. 19, 586–595.
37. Giraldez A.J., Cinalli R.M., Glasner, M.E., Enright, A.J., Thomson, J.M.,Baskerville, S., Hammond, S.M., Bartel, D.P., Schier, A.F.2005. MicroRNAs regulate brain morphogenesis in zebrafish. Science 308, 833–838.
38. Gocal G.F., Sheldon C.C., Gubler F., Moritz T., Bagnall D.J., MacMillan C.P., Li S.F., Parish R.W., Dennis E.S., Weigel D., King R.W. 2001. GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis. Plant Physiol. 127, 1682– 1693.
39. Gray W.M., Kepinski S., Rouse D., Leyser, O., Estelle M. 2001. Auxin regulates SCF (TIR1)-dependent degradation of AUX/IAA proteins. Nature 414, 271–276.
40. Gregory B.D., O Malley, R.C., Lister R., Urich M.A., Tonti-Filippini J., Chen H., Millar A.H., and Ecker J.R. 2008. A link between RNA metabolism and silencing affecting Arabidopsis development. Dev. Cell 14, 854–866.
41. Guo H.S., Xie Q., Fei J.F., Chua N.H., 2005. MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell 17, 1376– 1386.
42. Gurr S.J., Rushton P.J., 2005. Engineering plants with increased disease resistance: what are we going to express? Trends Biotechnol. 23, 275– 28
43. Hagen G., Guilfoyle T. 2002. Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Mol. Biol. 49, 373– 385.
44. Hammond S.M., Boettcher S., Caudy A.A., Kobayashi R., Hannon G.J. 2001. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146– 1150.
45. Hibara K., Takada S., Tasaka M., 2003. CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation. Plant J. 36, 687–696.
46. Hiei Y., Ohta S., Komari T. & Kumashiro T. 1994.Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271–282.
47. Iorio M.V., Ferracin M., Liu C.G., Veronese A., Spizzo R., Sabbioni S., Magri E., Pedriali M., Fabbri M., Campiglio M., Menard S., Palazzo J.P., Rosenberg A., Musiani P., Volinia S., Nenci I., Calin G.A., Jack T. 2004. Molecular and genetic mechanisms of floral control. Plant Cell 16,S1–S17.
48. Jones-Rhoades, M.W., Bartel D.P. 2004. Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol. Cell 14, 787– 799.
49. Juarez M.T., Kui J.S., Thomas J., Heller B.A., Timmermans M.C. 2004. microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity. Nature 428, 84–88.
50. Kan Nobuta, R C Venu, Cheng Lu, Andre′Belo′, Kalyan Vemaraju1, KarthikKulkarni, Wenzhong Wang, Manoj Pillay, Pamela J Green, Guo-liang Wang & Blake C Meyers. 2007. An expression atlas of rice mRNAs and small RNAs. NATURE BIOTECHNOLOGY 4,474-477
51. Kaneko M., Inukai Y., Ueguchi-Tanaka M., Itoh H., Izawa T., Kobayashi Y., Hattori T., Miyao A., Hirochika H., Ashikari M., Matsuoka M. 2004. Loss-of-function mutations of the rice GAMYB gene impair alpha-amylase expression in aleurone and flower development. Plant Cell 16, 33– 44.
52. Kasschau K.D., Xie Z., Allen E., Llave C., Chapman E.J., Krizan K.A., Carrington J.C. 2003. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. Dev. Cell 4, 205– 217.
53. Kidner C.A., Martienssen R.A., 2005. The developmental role of microRNA in plants. Curr. Opin. Plant Biol. 8, 38– 44.
54. Kim J., Jung J.H., Reyes J.L., Kim Y.S., Kim S.Y., Chung K.S., Kim J.A., Lee M., Lee Y., Kim V.N., Chua N.H., Park C.M., 2005. microRNAdirected cleavage of ATHB15 mRNA regulates vascular development in Arabidopsis inflorescence stems. Plant J. 42, 84–94.
55. Kim V.N. 2005. MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol. 6, 376–385.
56. Kurihara Y., Takashi Y., and Watanabe Y. 2006. The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12, 206–212.
57. Lagos-Quintana, M., Rauhut R., Lendeckel W., Tuschl T. 2001. Identification of novel genes coding for small expressed RNAs. Science 294, 853– 858.
58. Laubinger S., Sachsenberg T., Zeller G., Busch W., Lohmann J.U., Ratsch G., and Weigel D. 2008. Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 105, 8795–8800.
59. Laufs P., Peaucelle A., Morin H., Traas J. 2004. MicroRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems. Development 131, 4311–4322.
60. Lauter N., Kampani A., Carlson S., Goebel M., Moose S.P. 2005. MicroRNA172 down-regulates glossy15 to promote vegetative phase change in maize. Proc. Natl. Acad. Sci. U. S. A. 102, 9412– 9417.
61. Lee R.C., Ambros V. 2001. An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862– 864.
62. Li J., Yang, Z., Yu B., Liu J., and Chen, X. 2005. Methylation protects miRNAs and siRNAsfrom a 30-end uridylation activity in Arabidopsis. Curr. Biol. 15, 1501–1507.
63. Llave C., Xie Z., Kasschau K.D., Carrington J.C. 2002. Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 2053–2056.
64. Lohmann J.U., Weigel D. 2002. Building beauty: the genetic control of floral patterning. Dev. Cell 2, 135– 142.
65. Lu S., Sun Y.H., Shi R., Clark C., Li L., Chiang V.L., 2005. Novel and mechanical stress-responsive micrornas in Populus trichocarpa that are absent from Arabidopsis. Plant Cell 17, 2186– 2203.
66. Mallory A.C., Bartel D.P., Bartel B. 2005. MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17, 1360– 1375.
67. Mallory A.C., Dugas D.V., Bartel D.P., Bartel B., 2004b. MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs. Curr. Biol. 14, 1035–1046.
68. Mallory A.C., Reinhart B.J., Jones-Rhoades, M.W., Tang G., Zamore P.D., Barton M.K., Bartel D.P., 2004a. MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5V region. EMBO J. 23, 3356– 3364.
69. McConnell J.R., Emery J., Eshed Y., Bao N., Bowman J., Barton M.K. 2001. Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411, 709– 713.
70. McKhann H., Garcion C., Vaucheret H., Sandberg G., Bellini C. 2005. Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell 17, 1343– 1359.
71. Molnar A., Schwach F., Studholme D.J., Thuenemann E.C., and Baulcombe D.C. 2007. miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447, 1126–1129.
72. Ohashi-Ito K., Fukuda H. 2003. HD-Zip III homeobox genes that include a novel member, ZeHB-13 (Zinnia)/ATHB-15 (Arabidopsis), are Involved in procambium and xylem cell differentiation. Plant Cell Physiol. 44, 1350–1358.
73. Palatnik J.F., Allen E., Wu X., Schommer C., Schwab R., Carrington J.C., Weigel D. 2003. Control of leaf morphogenesis by microRNAs. Nature 425, 257–263.
74. Palme K., Hesse T., Moore I., Campos N., Feldwisch J., Garbers C., Hesse F., Schell J. 1991. Hormonal modulation of plant growth: the role of auxin perception. Mech. Dev. 33, 97– 106.
75. Pande S., Siddique K.H.M., Kishore G.K., Bayaa B., Gaur P.M., Gowda C.L.L., Bretag, T.W., Crouch J.H. 2005. Ascochyta blight of chickpea (Cicer arietinum L.): a review of biology, pathogenicity, and disease management. Aust. J. Agric. Res. 56, 317– 332.
76. Papp I., Mette M.F., Aufsatz W., Daxinger L., Schauer S.E., Ray A., van der Winden, J., Matzke M., Matzke A.J. 2003. Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiol. 132, 1382– 1390.
77. Park M.Y., Wu G., Gonzalez-Sulser A., Vaucheret H., and Poethig R.S. 2005. Nuclear processing and export of microRNAs in Arabidopsis. Proc. Natl. Acad. Sci. USA 102, 3691–3696.
78. Park W., Li J., Song R., Messing J., Chen X. 2002. CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12, 1484– 1495.
79. Peng Gao, Xi Bai,Liang Yang,De kang, Lv Yong, LiHua, CaiWei, JiDianjing, Guo Yanming Zhu. 2010. Over-expression of osa-MIR396c decreases salt and alkali stress tolerance. Planta 231,991–1001
80. Peng Gao, Xi Bai, et al.osa-MIR393: a salinity- and alkaline stress-related microRNA gene. Mol Biol Rep .2011.38:237–242
81. Piriyapongsa, J., and Jordan I.K. 2008. Dual coding of siRNAs and miRNAs by plant transposable elements. RNA 14, 814–821.
82. Plisson C., Drucker M., Blanc S., German-Retana S., Le Gall, O., Thomas D., Bron P. 2003. Structural characterization of HC-Pro, a plant virus multifunctional protein. J. Biol. Chem. 278, 23753– 23761
83. Qian-Hao Zhu, Andrew Spriggs, Louisa Matthew, et al. 2008. A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res. 18: 1456-1465
84. Qian-Hao Zhu, Narayana M Upadhyaya, Frank Gubler and Chris A Helliwell. 2009. Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa). BMC Plant Biology 149 1-13
85. Querzoli P., Negrini M., Croce C.M. 2005. MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 65, 7065– 7070.
86. Rajagopalan R., Vaucheret H., Trejo J., and Bartel D.P. 2006. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20 3407–3425.
87. Ramachandran V., and Chen X. 2008. Degradation of microRNAs by a family of exoribonucleases in Arabidopsis. Science 321, 1490–1492.
88. Ramanjulu Sunkar, Xuefeng Zhou, Yun Zheng, Weixiong Zhang and Jian-Kang Zhu, Reinhart B J, et al. 2002. MicroRNAs in plants. Genes and Development 6,1616-1626
89. Ramanjulu Sunkar, Xuefeng Zhou, Yun Zheng, Weixiong Zhang and Jian-Kang Zhu. 2008. Identification of novel and candidate miRNAs in rice by high throughput sequencing. BMC Plant Biology 8:25
90. Reinhart B.J., Weinstein E.G., Rhoades M.W., Bartel B., Bartel D.P. 2002. MicroRNAs in plants. Genes Dev. 16, 1616– 1626.
91. Rhoades M.W., Reinhart B.J., Lim L.P., Burge C.B., Bartel B., Bartel D.P. 2002. Prediction of plant microRNA targets. Cell 110, 513– 520.
92. Riechmann J.L., Heard J., Martin G., Reuber L., Jiang C., Keddie J., Adam L., Pineda O., Ratcliffe O.J., Samaha R.R., Creelman R., Pilgrim M., Broun P., Zhang J.Z., Ghandehari D.,Sherman B.K., Yu, G. 2000. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290, 2105– 2110.
93. Sam Griffiths-Jones, et al. 2006 miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research, 2005. 33(20): 179-187.
94. Schwab R., Palatnik J.F., Riester M., Schommer C., Schmid M., Weigel D. 2005. Specific effects of microRNAs on the plant transcriptome. Dev. Cell 8, 517– 527.
95. Si Qi Huang , Jie Peng , Cheng Xiang Qiu, Zhi Min Yang. 2009 Heavy metal-regulated new microRNAs from rice. Journal of Inorganic Biochemistry. 103, 282–287
96. Sorin C., Bussell J.D., Camus I., Ljung K., Kowalczyk M., Geiss G., McKhann, H., Garcion C., Vaucheret H., Sandberg G., Bellini C. 2005. Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell 17, 1343– 1359.
97. Sunkar R., Girke T., Zhu J.K. 2005. Identification and characterization of endogenous small interfering RNAs from rice. Nucleic Acids Res. 33, 4443–4454.
98. Sunkar R., Zhu J.K. 2004. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16, 2001– 2019.
99. Takada S., Hibara K., Ishida T., Tasaka M. 2000. The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation. Development 128, 1127– 1135.
100. Talmor-Neiman, M., Stav R., Frank W., Voss B., and Arazi T. 2006. Novel micro-RNAs and intermediates of micro-RNA biogenesis from moss. Plant J.47, 25–37.
101. Tian Li, Hui Li, Yun-Xiao Zhang and Jin-Yuan Liu. 2010 Identification and analysis of seven H2O2-responsive miRNAs and 32 new miRNAs in the seedlings of rice (Oryza sativa L. ssp. indica). Nucleic Acids Research, 2010, 1–13
102. Vaucheret H., Vazquez F., Crete P., Bartel D.P. 2004. The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 18, 1187– 1197.
103. Vazquez F., Blevins T., Ailhas J., Boller T., and Meins F., Jr. 2008. Evolution of Arabidopsis MIR genes generates novel microRNA classes. Nucleic Acids Res. 36, 6429–6438.
104. Vazquez F., Gasciolli V., Crete P., Vaucheret H. 2004. The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr. Biol. 14, 346– 351.
105. Weijers, D., Jurgens, G. 2005. Auxin and embryo axis formation: the ends in sight? Curr. Opin. Plant Biol. 8, 32– 37.
106. Wienholds E., Kloosterman W.P., Miska E., Alvarez-Saavedra E., Berezikov E., de Bruijn, E., Horvitz H.R., Kauppinen S., Plasterk R.H. 2005. MicroRNA expression in zebrafish embryonic development. Science 309, 310– 311.
107. Woodward A.W., Bartel B. 2005. Auxin: Regulation, action, and interaction. Ann. Bot. 95, 707– 735.
108. Xie Z., Kasschau, K.D., Carrington J.C. 2003. Negative feedback regulation of Dicer-Like1 inArabidopsis by microRNA-guided mRNA degradation. Curr. Biol. 13, 784–789.
109. Yan K.S., Yan S., Farooq A., Han A., Zeng L., Zhou M.M. 2003. Structure and conserved RNA binding of the PAZ domain. Nature 426, 468– 474.
110. Yang Z., Ebright Y.W., Yu B., and Chen X. 2006. HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 20 OH of the 30 terminal nucleotide. Nucleic Acids Res. 34, 667–675.
111. Yu B., Bi L., Zheng B., Ji L., Chevalier D., Agarwal M., Ramachandran V., Li W., Lagrange T., Walker J.C., and Chen X. 2008. The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc. Natl. Acad. Sci. USA 105, 10073–10078.
112. Zhang B., Pan X., and Stellwag E.J. 2008. Identification of soybean micro-RNAs and their targets. Planta 229, 161–182.
113. Zhang B., Pan X., Cannon C.H., Cobb G.P., and Anderson, T.A. 2006. Conservation and divergence of plant microRNA genes. Plant J. 46, 243–259.
114. Zhang B.H., Liu F., Yao C.B., Wang K.B. 2000. Recent progress in cotton biotechnology and genetic engineering in China. Curr. Sci. 79, 37–44.
115. Zhang B.H., Pan X.P., Wang Q.L., Cobb G.P., Anderson T.A. 2005. Identification and characterization of new plant microRNAs using EST analysis. Cell Res. 15, 336– 360.
116. Zhong R., Ye Z.H. 2004. Amphivasal vascular bundle 1, a gain-offunction mutation of the IFL1/REV gene, is associated with alterations in the polarity of leaves, stems and carpels. Plant Cell Physiol. 45, 369– 385.