Molecular cloning, expression profile and subcellular localization of nanos1 gene from Macrobrachium rosenbergii
-
摘要: 为探究nanos1基因在罗氏沼虾 (Macrobrachium rosenbergii) 生殖发育中的功能，克隆了罗氏沼虾nanos1基因，其cDNA序列全长2 811 bp，编码243个氨基酸；系统进化分析结果表明其与中华绒螯蟹 (Eriocheir sinensis) 的同源性最高，与鱼类的nanos1同源性较高，与哺乳类同源性较低。半定量PCR检测发现，nanos1在卵巢中特异表达。通过实时荧光定量PCR检测该基因在不同胚胎发育时期及幼体中的表达水平，结果显示：nanso1 mRNA在 (卵) 未受精时期表达量最高，显著高于 (卵) 受精期及胚胎发育各期 (P<0.05)；在胚胎发育期间， (卵) 受精时期表达量最高，显著高于卵裂期且极显著高于胚胎发育后期 (P<0.01)；该基因在卵裂期的表达水平显著高于囊胚期至幼体期；而囊胚期至幼体期之间表达水平较低且无差异。原位杂交技术检测显示，nanos1 mRNA在卵原细胞及初级卵母细胞 (Oc1，Oc2，Oc3，Oc4) 的细胞质中表达。以上结果表明，nanos1与罗氏沼虾雌性生殖细胞发育有着密切的关系。Abstract: To investigate the function of the nanos1 gene in the reproductive development of Macrobrachium rosenbergii, we cloned its nanos1 gene. The cDNA sequence was 2 811 bp, encoding 243 amino acids. Phylogenetic analysis shows that it had the highest homology with Eriocheir sinensis, higher homology with nanos1 in fish but lower homology with mammals. We detected the expression level of the gene at different embryonic developmental stages and in larvae by real-time fluorescence quantitative PCR. The results show that the expression level of nanso1 mRNA was the highest at unfertilized stage, significantly higher than that at fertilized stage and the other embryonic developmental stages (P<0.05). During the embryonic development, the expression was the highest at fertilization stage, significantly higher than that at cleavage stage and very significantly higher than that at late embryonic development (P<0.01). The expression level of this gene at cleavage stage was significantly higher than that from blastocyst stage to larval stage. The expression level from blastocyst stage to larval stage was low and there was no difference.In situ hybridization shows that nanos1 mRNA was expressed in the cytoplasm of oocytes and primary oocytes (Oc1, Oc2, Oc3 and Oc4). The results show that nanos1 is closely related to the development of female germ cells of M. rosenbergii.
-
Key words:
- Macrobrachium rosenbergii /
- nanos1 /
- Expression profile /
- Subcellular localization
-
图 1 罗氏沼虾Mrnanos1 cDNA序列及氨基酸序列
Figure 1. Sequence and amino acid sequence ofMrnanos1 cDNA from M. rosenbergii
图 2 MrNanos1氨基酸序列与其他物种Nanos家族蛋白的系统进化树分析
Figure 2. Phylogenetic tree analysis of MrNanos1 amino acid sequence with Nanos family proteins of other species
图 3 罗氏沼虾MrNanos1与其他物种锌指区域结构的比较
Figure 3. Multiple alignments analysis of zinc finger domain of MrNanos1 in M. rosenbergii and other species
图 4 Mrnanos1基因在罗氏沼虾不同组织中的表达分析
Figure 4. Expression analysis of Mrnanos1 gene in different tissues of M. rosenbergii
图 5 罗氏沼虾nanos1在不同胚胎发育时期及早期幼体中的相对表达量
注：字母不同表示同一时间各实验组之间存在显著性差异 (P<0.05)。
Figure 5. Relative expression level ofnanos1 in different embryonic develomental stages and larvae of M. rosenbergii
Note: Different letters indicate significant difference among the experimental groups (P<0.05).
图 6 Mrnanos1 mRNA 在罗氏沼虾卵巢中的亚细胞分布
注：a. 罗氏沼虾卵巢切片 (HE 染色) ；b. 罗氏沼虾卵巢切片 (反义探针结果)；c. 罗氏沼虾卵巢切片 (正义探针结果)；d、e、f 分别为 a、b、c 中红色方框内的放大视野图。
Figure 6. Subcellular localization of Mrnanos1 mRNA in ovary of M. rosenbergii
Note: a. Ovarian sections of M. rosenbergii (HE staining); b. Ovarian sections of M. rosenbergii (Antisense probe results); c. Ovarian sections of M. rosenbergii (Results of sense probe); d, e and f. The enlarged view in the red box of Fig. a, b and c, respectively.
表 1 本研究所用的全部引物
Table 1. All primers used in this study
引物
Primer序列 (5'—3')
Sequence (5'–3')长度
Length/bp温度
Temperature/℃应用
Applicationnanos1 F
nanos1 RAAAACAACTAACGGCGATGC
ACGAAGACCCGCTGATATTG747 57 RT-PCR、PCR、原位杂交 nanos1 QF
nanos1 QRGGCCTTAGGGCAAATAGCTC
ACGAAGACCCGCTGATATTG233 60 qPCR β-actinF
β-actinRGAAGCGTACATGGTGGGACT
GGTGACCTGCGAGATTCATT466 57 RT-PCR β-actinQF
β-actinQRCAGGGAAAAGATGACCCAGA
GGAAGTGCATACCCCTCGTA161 60 qPCR 
下载: 导出CSV
-
[1] 肖楚康, 方刘, 阮国良, 等. 罗氏沼虾淡化养殖的现状与展望[J]. 江苏农业科学, 2019(8): 29-33. [2] 俞炎琴. 罗氏沼虾中性别发育相关基因Sxl和Dmrt基因的分子特征和功能研究[D]. 杭州: 浙江大学, 2013: 11- 13. [3] 姜建萍, 袁翔, 邱庆庆, 等. 罗氏沼虾性别相关基因研究进展及其单性化养殖现状[J]. 南方农业学报 2019, 50(9): 2111-2118. [4] SAGI A, AFELAO E D. The androgenic gland and monosex culture of freshwater prawn Macrobrachium rosenbergii (de Man): a biotechnological perspective[J]. Aquac Res, 2005, 36(3): 231-237. doi: 10.1111/j.1365-2109.2005.01238.x [5] SAGI A, SNIR E, KHALAILA I. Sexual differentiation in decapod crustaceans: role of the androgenic gland[J]. Invertebr Reprod Dev, 1997, 31(1/2/3): 55-61. [6] 彭巧玲, 浦友光, 程子华, 等. 罗氏沼虾3个Dmrt基因的序列分析[J]. 中国水产科学, 2005, 12(1): 5-9. doi: 10.3321/j.issn:1005-8737.2005.01.002 [7] ESCRIVA H, DELAUNAY F, LAUDET V. Ligand binding and nuclear receptor evolution[J]. Bioessays, 2000, 22(8): 717-727. doi: 10.1002/1521-1878(200008)22:8<717::AID-BIES5>3.0.CO;2-I [8] NAKKRASAE L I, DAMRONGPHOL P. A vasa-like gene in the giant freshwater prawn, Macrobrachium rosenbergii[J]. Mol Reprod Dev, 2010, 74(7): 835-842. [9] NGEROUNGNER A, NGEROUNGNER P, KAVANAUGH S, et al. The identification and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the giant freshwater prawn, Macrobrachium rosenbergii[J]. Invertebr Neurosci, 2008, 8(1): 49-57. doi: 10.1007/s10158-008-0067-5 [10] LUGO J M, MORERA Y, TANIA R et al. Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti. Functional analysis by double-stranded RNA interference technique[J]. Febs J, 2010, 273(24): 5669-5677. [11] ROSEN O, MANOR R, SAGI A, et al. A sexual shift induced by silencing of a single insulin-like gene in crayfish: ovarian up-regulation and testicular degeneration[J]. PLoS One, 2010, 5(12): e15281. doi: 10.1371/journal.pone.0015281 [12] VENTURA T, MANOR R, AFLALO E D, et al. Timing sexual differentiation: full function sex reversal achieved through silencing of a single insulin-like gene in the prawn, Macrobrachium rosenbergii[J]. Biol Reprod, 2012, 86(3): 90-103. [13] 周林燕, 张修月, 王德寿. 脊椎动物性别决定和分化的分子机制研究进展[J]. 动物学研究, 2004, 25(1): 81-88. doi: 10.3321/j.issn:0254-5853.2004.01.015 [14] 杨莹莹, 王浩栋, 张艺元, 等. 性别决定相关因子的研究进展[J]. 现代生物医学进展, 2018, 18(1): 168-174. [15] ZHONG W, LIN H. Nanos maintains germline stem cell self-renewal by preventing differentiation[J]. Science, 2004, 303(5666): 2016-2019. doi: 10.1126/science.1093983 [16] ASAOK T M, YAMADA M, NAKAMURA A, et al. Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos[J]. Nat Cell Biol, 1999, 1(7): 431-437. doi: 10.1038/15666 [17] SUBRAMANIAM K, SEYDOUX G. nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans[J]. Development, 1999, 126(21): 4861-4871. doi: 10.1242/dev.126.21.4861 [18] KRAEMER B, CRITTENDEN S, GALLEGOS M, et al. NANOS-3 and FBF proteins physically interact to control the sperm-oocyte switch in Caenorhabditis elegans[J]. Curr Biol, 1999, 9(18): 1009-1018. doi: 10.1016/S0960-9822(99)80449-7 [19] TSUDA M, SASAOKA Y, KISO M, et al. Conserved role of nanos proteins in germ cell development[J]. Science, 2003, 301(5637): 1239-1241. doi: 10.1126/science.1085222 [20] ANDRIES V, KEUCKELAERE E D, STAES K, et al. A new mouse model to study the role of ectopic Nanos3 expression in cancer[J]. BMC Cancer, 2019, 19(1): 598-606. doi: 10.1186/s12885-019-5807-x [21] EXTAVOUR C G, PANG K, MAYUS D Q, et al. vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms[J]. Evol Develop, 2005, 7(3): 201-215. doi: 10.1111/j.1525-142X.2005.05023.x [22] MOCHIZUKI K, SANO H, KOBAYASHI S, et al. Expression and evolutionary conservation of nanos-related genes in Hydra[J]. Develop Genes Evol, 2000, 210(12): 591-602. doi: 10.1007/s004270000105 [23] 刘俊卿, 孙敏, 王兰. 河南华溪蟹nanos1、nanos2、piwi基因部分序列的克隆及表达特征[J]. 淡水渔业, 2020, 50(5): 10-16. doi: 10.3969/j.issn.1000-6907.2020.05.002 [24] YU L, XU D, YE H, et al. Gonadal transcriptome analysis of Pacific abaloneHaliotis discus: identification of genes involved in germ cell development[J]. Mar Biotechnol, 2018, 20(5): 1-14. [25] YE H, CHEN X, WEI Q, et al. Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis[J]. Gene, 2012, 511: 285-292. doi: 10.1016/j.gene.2012.09.005 [26] HARAGUCHI S, TSUDA M, KITAJIMA S, et al. nanos1 a mouse nanos gene expressed in the central nervous system is dispensable for normal development[J]. Mech Develop, 2003, 120(6): 721-731. doi: 10.1016/S0925-4773(03)00043-1 [27] JARUZELSKA J, KOTECKI M, KUSZ K, et al. Conservation of a Pumilio-Nanos complex from Drosophila germ plasm to human germ cells[J]. Develop Genes Evol, 2003, 213(3): 120-126. doi: 10.1007/s00427-003-0303-2 [28] KEUCKELAERE E D, HULPIAU P, SAEYS Y, et al. Nanos genes and their role in development and beyond[J]. Cell Mol Life Sci, 2018, 75(6217): 1-18. [29] LAI F F, SINGH A, KING M L. Xenopus Nanos1 is required to prevent endoderm gene expression and apoptosis in primordial germ cells[J]. Development, 2012, 139(8): 1476-1486. doi: 10.1242/dev.079608 [30] ZHAO G, CHEN K, YAO Q, et al. The nanos gene of Bombyx mori and its expression patterns in developmental embryos and larvae tissues[J]. Gene Expr Patterns, 2008, 8: 254-260. doi: 10.1016/j.gep.2007.12.006 [31] WU H R, CHEN Y T, SU Y H, et al. Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae[J]. Develop Biol, 2011, 353(1): 147-159. doi: 10.1016/j.ydbio.2011.02.014 [32] NAKO H, MATASUMOTO T, OBA Y, et al. Germ cell specification and early embryonic patterning in Bombyx mori as revealed by nanos orthologues[J]. Evol Develop, 2010, 10(5): 546-554. [33] DEARDEN P K. Germ cell development in the Honeybee (Apis mellifera); Vasa and Nanos expression[J]. BMC Develop Biol, 2006, 6(1): 6. doi: 10.1186/1471-213X-6-6 [34] MARION K, THISSE C, THISSE B, et al. A zebrafish nanos-related gene is essential for the development of primordial germ cells[J]. Genes Dev, 2001, 15(21): 2877-2885. doi: 10.1101/gad.212401 [35] JANECKI D M, ILASLAN E, SAMIALEK M J, et al. Human NANOS1 represses apoptosis by downregulating pro-apoptotic genes in the male germ cell line[J]. Int J Mol Sci, 2020, 21(8): 3009. doi: 10.3390/ijms21083009 [36] LIU Q, ZHU L L, LIU F C, et al. Function of Nanos1 gene in the development of reproductive organs of Schistosoma japonicum[J]. Parasitol Res, 2017, 116(5): 1505-1513. doi: 10.1007/s00436-017-5427-9 -
下载:
点击查看大图
计量
- 文章访问数: 565
- HTML全文浏览量: 128
- PDF下载量: 24
- 被引次数: 0
粤公网安备 44010502001741号
