Side-by-side Chinese-English

家蚕let-7 microRNA靶基因Bmlin-41的克隆表达与调控

周兰庭1,2 周挺1 高君凌1 王伟1 吴小燕1 黄亚玺1 夏庆友1 刘仕平1

(1.西南大学家蚕基因组生物学国家重点实验室, 重庆 400716)
(2.湖北文理学院医学院, 湖北襄阳 441053)

【摘要】异时性基因调控细胞增殖和个体发育阶段的转换。家蚕异时性基因在家蚕变态发育过程中也很可能具有重要的调控作用,但它们的表达模式、生物学功能以及与micro RNA之间的关系却鲜有报道。本研究首先利用果蝇同源基因lin-41搜索家蚕基因组数据库中相似序列,设计引物扩增Bmlin-41的编码序列,克隆了家蚕Bmlin-41基因CDS,其长度为2 166 bp,编码721个氨基酸,含有B-box和NHL结构域;随后,利用RT-PCR、q PCR技术并结合已有的家蚕全基因组芯片数据研究了Bmlin-41在家蚕中的时空表达模式,发现Bmlin-41在从家蚕胚胎到成虫的发育过程中呈逐渐递增的表达趋势,在五龄3 d不同组织中,于卵巢里表达量最高,精巢和中肠次之,而其余组织中低量表达或不表达;最后,利用3′RACE克隆了Bmlin-41基因的3′UTR,全长1 434 bp,用在线软件RNAhybrid预测发现Bmlin-41的3′UTR上存在bmo-let-7靶位点,构建了含Bmlin-41 3′UTR的双荧光素酶报告基因载体,在S2细胞上共转染Bmlin-41 3′UTR和bmo-let-7的模拟物(Mimics)和拮抗剂(Antagomir),bmo-let-7 mimics显著下调Bmlin-41,bmo-let-7 antagomir显著上调Bmlin-41,证实了Bmlin-41是bmo-let-7的靶基因。以上研究结果为深入研究let-7 mi RNA和Bmlin-41的功能,揭示Bmlin-41和bmo-let-7在家蚕变态发育过程中的调控关系提供了新的线索。

【关键词】 家蚕;异时性基因;家蚕let-7 miRNA;Bmlin-41;表达模式;靶基因;

【DOI】

【基金资助】 国家重点基础研究发展计划(973计划)(No.2012CB114602); 国家自然科学基金(Nos.31071136,31571334); 重庆市基础与前沿研究计划(No.cstc2014jcyjA00025)资助;

Cloning and expression profile of Bmlin-41 and its regulation by the silkworm microRNA let-7

Lanting Zhou1,2 Ting Zhou1 Junling Gao1 Wei Wang1 Xiaoyan Wu1 Yaxi Huang1 Qingyou Xia1 Shiping Liu1

(1.State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China 400716)
(2.Medical College, Hubei University of Arts and Science, Xiangyang, Hubei, China 441053)

【Abstract】The heterochronic genes regulate cell proliferation and switch development stage transitions. Heterochronic genes might also play important roles in regulating the development of silkworm, but very few of their expression profiles, functions and their relationship with micro RNAs are available so far. Firstly, in this work, the primers for cloning Bmlin-41 were designed based on the homologous sequence of known Drosophila melanogaster lin-41, which was used as the query to blast against SilkDB. The obtained full CDS (2 166 bp) of Bmlin-41 encodes 721 amino acids and contains B-box and NHL domains. Then, the spatiotemporal expression patterns of Bmlin-41 were characterized by RT-PCR, quantitative real time PCR as well as our lab's previous silkworm genome microarray data. Bmlin-41 was increasingly expressed from embryonic to adult stage. In diverse tissues of day-3 fifth instar larvae, Bmlin-41 showed the highest accumulation in ovary, secondly in testis and midgut, but very low expression was observed in other tissues. Finally, 3′UTR of Bmlin-41 of 1 434 bp in length was cloned by rapid-amplification of cDNA ends (3'RACE) and was predicted to bare two binding sites of bmo-let-7 by using online RNAhybrid. To verify the binding effect, 3′UTR was cloned into psi-CHECK-2 vector and submitted to dual luciferase assay in the S2 cells in vitro. The dual luciferase assay demonstrated that Bmlin-41 was down-regulated by bmo-let-7 mimics and upregulated by bmo-let-7 antagomir, thus confirming the Bmlin-41 is negatively regulated by bmo-let-7. Our work might help further study on the roles of Bmlin-41 and bmo-let-7 and their regulation relationship involved in controlling metamorphosis of silkworm.

【Keywords】 silkworm (Bombyx mori); heterochronic gene; bmo-let-7 miRNA; Bmlin-41; expression profile; target gene;

【DOI】

【Funds】 National Basic Research Program of China (973 Program) (No. 2012CB114602); National Natural Science Foundation of China (Nos. 31071136, 1571334); Fundamental and Advanced Research Projects of Chongqing (No. cstc2014jcyjA00025);

Download this article
    References

    [1] Ambros V. A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans. Cell, 1989, 57(1):49–57.

    [2] Ambros V, Horvitz HR. Heterochronic mutants of the nematode Caenorhabditis elegans. Science, 1984, 226(4673):409–416.

    [3]Ambros V, Horvitz HR. The lin-14 locus of Caenorhabditis elegans controls the time of expression of specific postembryonic developmental events. Genes Dev, 1987, 1(4):398–414.

    [4]Ambros V, Moss EG. Heterochronic genes and the temporal control of C. elegans development. Trends Genet, 1994, 10(4):123–127.

    [5]Slack F, Ruvkun G. Temporal pattern formation by heterochronic genes. Annu Rev Genet, 1997, 31:611–634.

    [6]Bettinger JC, Lee K, Rougvie AE. Stage-specific accumulation of the terminal differentiation factor LIN-29 during Caenorhabditis elegans development. Development, 1996, 122(8):2517–2527.

    [7]Slack FJ, Basson M, Liu ZC, et al. The lin-41RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol Cell, 2000, 5(4):659–669.

    [8]Loedige I, Gaidatzis D, Sack R, et al. The mammalian TRIM-NHL protein TRIM71/LIN-41is a repressor of m RNA function. Nucleic Acids Res, 2013, 41(1):518-532.

    [9]Ecsedi M, Großhans H. LIN-41/TRIM71:emancipation of a mi RNA target. Genes Dev, 2013, 27(6):581–589.

    [10]O'Farrell F, Esfahani SS, Engström Y, et al. Regulation of the Drosophila lin-41 homologue dappled by let-7 reveals conservation of a regulatory mechanism within the LIN-41 subclade. Dev Dyn, 2008, 237(1):196–208.

    [11]Spike, C. A, Coetzee, D, Eichten, C, et al. The TRIM-NHL protein LIN-41 and the OMA RNA-binding proteins antagonistically control the prophase-to-metaphase transition and growth of Caenorhabditis elegans oocytes. Genetics, 2014, 198(4):1535-1558.

    [12]Rybak A, Fuchs H, Hadian K, et al. The let-7target gene mouse lin-41 is a stem cell specific E3ubiquitin ligase for the mi RNA pathway protein Ago2. Nat Cell Biol, 2009, 11(12):1411–1420.

    [13]Tocchini C, Keusch JJ, Miller SB, et al. The TRIM-NHL protein LIN-41 controls the onset of developmental plasticity in Caenorhabditis elegans. PLo S Genet, 2014, 10(8):e1004533.

    [14]Ecsedi M, Rausch M, Großhans H. The let-7micro RNA directs vulval development through a single target. Dev Cell, 2015, 32(3):335–344.

    [15]Bartel DP. Micro RNAs:genomics, biogenesis, mechanism, and function. Cell, 2004, 116(2):281–297.

    [16]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 1993, 75(5):843–854.

    [17]Arasu P, Wightman B, Ruvkun G. Temporal regulation of lin-14 by the antagonistic action of two other heterochronic genes, lin-4 and lin-28. Genes Dev, 1991, 5(10):1825–1833.

    [18]Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993, 75(5):855–862.

    [19]Reinhart BJ, Slack FJ, Basson M, et al. The21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature, 2000, 403(6772):901–906.

    [20]Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature, 2000, 408(6808):86–89.

    [21]Vella MC, Choi EY, Lin SY, et al. The C. elegans micro RNA let-7 binds to imperfect let-7complementary sites from the lin-41 3'UTR. Genes Dev, 2004, 18(2):132–137.

    [22]Kloosterman WP, Wienholds E, Ketting RF, et al. Substrate requirements for let-7 function in the developing zebrafish embryo. Nucleic Acids Res, 2004, 32(21):6284–6291.

    [23]Kanamoto T, Terada K, Yoshikawa H, et al. Cloning and regulation of the vertebrate homologue of lin-41 that functions as a heterochronic gene in Caenorhabditis elegans. Dev Dyn, 2006, 235(4):1142–1149.

    [24]Abbott AL, Alvarez-Saavedra E, Miska EA, et al. The let-7 Micro RNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans. Dev Cell, 2005, 9(3):403–414.

    [25]Esquela-Kerscher A, Johnson SM, Bai L, et al. Post-embryonic expression of C. elegans micro RNAs belonging to the lin-4 and let-7 families in the hypodermis and the reproductive system. Dev Dyn, 2005, 234(4):868–877.

    [26]Großhans H, Johnson T, Reinert KL, et al. The temporal patterning micro RNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans. Dev Cell, 2005, 8(3):321–330.

    [27]Alvarez-Garcia I, Miska EA. Micro RNA functions in animal development and human disease. Development, 2005, 132(21):4653–4662.

    [28]Ambros V. The functions of animal micro RNAs. Nature, 2004, 431(7006):350–355.

    [29]Lujambio A, Ropero S, Ballestar E, et al. Genetic unmasking of an epigenetically silenced micro RNA in human cancer cells. Cancer Res, 2007, 67(4):1424–1429.

    [30]Brueckner B, Stresemann C, Kuner R, et al. The human let-7a-3 locus contains an epigenetically regulated micro RNA gene with oncogenic function. Cancer Res, 2007, 67(4):1419–1423.

    [31]Liu SP, Xia QY, Zhao P, et al. Characterization and expression patterns of let-7 micro RNA in the silkworm(Bombyx mori). BMC Dev Biol, 2007, 7(1):88.

    [32]Ling L, Ge X, Li ZQ, et al. Micro RNA Let-7regulates molting and metamorphosis in the silkworm, Bombyx mori. Insect Biochem Mol Biol, 2014, 53:13–21.

    [33]Liu SP, Xia QY. Protocol of Northern blotting hybridization for micro RNA detection of silkworm (Bombyx mori). Sci Sericul, 2014, 40(4):724–729 (in Chinese).

    [34]Higgins DG, Thompson JD, Gibson TJ. Using CLUSTAL for multiple sequence alignments. Methods Enzymol, 1996, 266:383–402.

    [35]Tamura K, Stecher G, Peterson D, et al. MEGA 6. 0:molecular evolutionary genetics analysis version6. 0. Mol Biol Evol, 2013, 30(12):2725–2729.

    [36]Xia QY, Cheng DJ, Duan J, et al. Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori. Genome Biol, 2007, 8(8):R162.

    [37]Kozomara A, Griffiths-Jones S. mi RBase:annotating high confidence micro RNAs using deep sequencing data. Nucleic Acids Res, 2014, 42:D68–D73.

    [38]Rehmsmeier M, Steffen P, Höchsmann M, et al. Fast and effective prediction of micro RNA/target duplexes. RNA, 2004, 10(10):1507–1517.

    [39]Letunic I, Doerks T, Bork P. SMART:recent updates, new developments and status in 2015. Nucleic Acids Res, 2015, 43:D257–D260.

    [40]Zhou T. Cloning of let-7 target genes and inducing of ecdysone on bmo-let-7 mi RNA in silkworm cell lines[D]. Chongqing:Southwest University, 2009 (in Chinese).

    [41]Sempere LF, Dubrovsky EB, Dubrovskaya VA, et al. The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster. Dev Biol, 2002, 244(1):170–179.

    [42]Liu SP, Huang YX, Yin JY, et al. Cloning and expression profile of Bmyan in the silkworm (Bombyx mori) and experimental validation as one target of micro RNA 7. Chin J Biotech, 2015, 31(11):1612–1622(in Chinese).

This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 05, Pages 635-647

May 2016

Downloads:2

Share
Article Outline

Knowledge

Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion
  • 4 Conclusions
  • References