Freshwater Fisheries Jan gdf11 cdna Acipenser dabryanus gdf11 growth differentiation factor 11 cdna gdf11 cdna 1 2

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1 Freshwater Fisheries Jan gdf11 cdna Acipenser dabryanus gdf11 growth differentiation factor 11 cdna gdf11 cdna bp Poly A bp bp 3 88 bp Signal P N 21aa gdf11 7 gdf11 gdf ± 3. 0 cm /s gdf11 Acipenser dabryanus gdf11 S DOI: /j.cnki.dsyy A Cloning and expression pattern of gdf11 cdna in Acipenser dabryanus DU Hao LENG Xiao-qian ZHANG Shu-huan YE Huan SHEN Li LIU Zhi-gang CHEN Xi-hua WEI Qi-wei Yangtze River Fisheries Research Institute Chinese Academy of Fishery Science Key Laboratory of Freshwater Biodiversity Conservation Ministry of Agriculture Wuhan China Abstract The full - length cdna of the gdf11 growth differentiation factor 11 gene from Acipenser dabryanus was cloned. The gdf11 gene cdna is bp in size and has a bp ORF open reading frame encoding a putative protein of 396 amino acid residues and 5 and 3 non - coding region were 19 bp and 88 bp respectively. By Signal P prediction it was found that A. dabryanus gdf11 protein contained a 21 amino acid signal peptide. The RT - qpcr analysis showed that gdf11 was existed in all the examined tissues and it was expressed highly in the eyes and gills. Under different flow conditions significant differences were found in the gdf11 expression level of the gills and heart the expression of gdf11 gene in the gill and heart of A. dabryanus in the static water group is times and times of those in the flow enriched water group ± 3. 0 cm /s. These implied gdf11 might have functions in the respiratory metabolism. Key words Acipenser dabryanus gdf11 gene Gene cloning Tissue expression pattern gdf11 growth differentiation factor β Transforming Growth Factor β superfamily TGF - β gdf11 4 Sparus aurata gdf11 Mcpherron 1 gdf11 Gamer 2 5 gdf11 gdf11 Acipenser dabryanus Danio rerio gdf weiqw@ yfi. ac. cn duhao@ yfi. ac. cn

2 1 gdf11 cdna Clustal X2 10 Primer Premier 5. 0 Oligo 6. 0 dead end 11 vasa 12 1 SMART cdna gdf11 cdna PCR peggy 3 gdf11 gdf11 gdf11 1 SMART cdna gdf11 RACE 5 3RACE DNAstar 2 gdf11 cdna BLAST ORF Finder - 80 ExPASy ± 3. 0 cm /s 8 Molecular Biology Server DNA 1. 2 RNA SMART cdna RNA RNeasy Plus Mini Kit Qiagen cdna PrimeScript RT reagent Kit With gdna Eraser RNA Super SMART PCR R β - actin cdna Synthesis Kit 30 s s 77 SMART cdna gdf11 GenBank gdf11 1% 1. 4 Clustal W 1. 5 gdf11 gdf11 PCR Adgdf11 - qrt - F Adgdf11 - qrt - cdna real - time PCR PCR 94 3 min s 57 5 s ~ gdf11 Tab. 1 Sequences of the primers used for gdf11 gene 5'- 3' Degenerate_ forward Partial GCYCCNAAYATYAGCMGRGA Degenerate_ reverse Partial VAGCATGTTGATGGGNGACA Adgdf11-3RACE forward RACE ACTGCGACGAGCACTCCCTGGA Adgdf11-3RACE nested forward RACE CTGCTCCGGCCAGTGCGAGTAC Adgdf11-5RACE reverse RACE GGACAAGGGGCTCCGGTTCTGA Adgdf11-5RACE nested reverse RACE CTGGAGCGGAGGGGCCTTG Adgdf11 - qf RT - PCR CTGCTTCTTCAAGTTCAACCCCAA Adgdf11 - qr RT - PCR GAGGTCACTGCCAGGTCGTTGC Adβ - actin - F Control TTCACCACCACAGCCGAGAG Adβ - actin - R Control AGGTCCTTACGGATGTCAACG 3 - AP RACE GGTATCAACGCAGAGTACTT 5 - AP RACE ATCAACGCAGAGTACGCGGG

gdf11 基因特异引物通过 3ＲACE PCＲ的方法获得了达氏鲟 gdf11 基因 cdna 序列的 3 端共 315 bp 通过 5ＲACE PCＲ获得了基因 cdna 序列的 5 端共 343 bp 图2 通过序列拼接得到了达氏鲟 gdf11 基因的全长 cdna 序列

2 达氏鲟 gdf11 基因 cdna 序列特征达氏鲟 gdf11 基因的 cdna 序列全长 1 298 bp 不包括 Poly A 开放阅读框为 1 191 bp 编码图3 Fig. 3 图2 达氏鲟 gdf11 基因 3 端和 5 端 cdna 序列的克隆 Fig.

3 淡 32 水 2 结果 2. 1 达氏鲟 gdf11 基因全长 cdna 的克隆根据 gdf11 基因家族保守的区域设计简并引物以达氏鲟脑 SMAＲT ds cdna 为模板 PCＲ扩增得到了一个大小为 851 bp 的片段图1 将该片渔业 2017 年证明为 gdf11 基因然后根据扩增片段的序列设计 gdf11 基因特异引物通过 3ＲACE PCＲ的方法获得了达氏鲟 gdf11 基因 cdna 序列的 3 端共 315 bp 通过 5ＲACE PCＲ获得了基因 cdna 序列的 5 端共 343 bp 图2 通过序列拼接得到了达氏鲟 gdf11 基因的全长 cdna 序列段克隆测序后通过 BLAST 进行同源搜索结果图1 Fig. 1 达氏鲟 gdf11 基因保守片段的克隆 Cloning of the conserved fragment of gdf11 gene in A. dabryanus M DNA Marker DL 达氏鲟 gdf11 基因 cdna 序列特征达氏鲟 gdf11 基因的 cdna 序列全长 bp 不包括 Poly A 开放阅读框为 bp 编码图3 Fig. 3 图2 达氏鲟 gdf11 基因 3 端和 5 端 cdna 序列的克隆 Fig. 2 Cloning of the 3 and 5 end sequence of gdf11 gene in A. dabryanus M DNA Marker DL 个氨基酸 5 非编码区长 19 bp 3 非编码区长 88 bp 图3 通过 Signal P 软件预测含有 N 端 21aa 的信号肽达氏鲟 gdf11 基因全长 cdna 序列及其推导的氨基酸序列 Nucleotide sequence of gdf11 in A. dabryanus and its deduced amino acids sequence. 下划线的氨基酸为信号肽起始密码子终止密码子和加尾信号为粗体

4 1 gdf11 cdna gdf11 gdf11 NCBI Ensembl gdf11 88% gdf11 88% 88% 87% RSPP 87% 86% 86% % 70% 70% 4 gdf11 Fig. 4 Multiple amino acids alignments of the gdf gdf11 gdf11 β - actin Real - time gdf11 PCR gdf11 8 cdna

5 aa gdf11 C 90% 9 gdf11 gdf11 gdf11 5 gdf11 Fig. 5 Analysis of the expression pattern of gdf11 in gdf11 gdf11 A. dabryanus P < gdf11 mrna gdf gdf11 gdf11 mrna 6 gdf11 mrna gdf11 mrna gdf11 gdf11 mrna gdf11 mrna gdf11 gdf11 3 C V gdf11 cdna gdf11 gdf11 N 6 gdf11 1 McPherron A C Lawler A M Lee S J. Regulation of anterior /posterior mrna patterning of the axial skeleton by growth / differentiation factor Fig. 6 Differential expression of Adgdf11 in static and flowing water 11 J. Nat Genet Gamer L W Wolfman N M Celeste A J et al. A novel BMP expressed in developing mouse limb spinal cord and tail bud is a potent mesoderm inducer in Xenopus embryos J. Dev Biol Peggy R Steven B Dimitar B et al. The isolation characterization and expression of a novel gdf11 gene and a second myostatin form in zebrafish Danio rerio J. Comp Biochem Physiol b - Biochem Mol Biol

6 Botstein D White R L Skolnick M et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms J. Am J Hum Genet Wright S. Evolution and the genetics of populations M. Varia- 23. M. bility within and among natural populations of Chicago Press Sophie Hubert D H. Linkage maps of microsatellite DNA markers for the pacific oyster crassostrea gigas J. Genetics Cordeiro G M Maguire T L Edwards K J et al. Optimisation of a microsatellite enrichment technique in Saccharum spp J. Plant Mol Biol Rep Thorpe J P. The molecular clock hypothesis biochemical evolution genetic differentiation and systematics J. Annual Rev Ecol Systematics Sunnucks P. Efficient genetic markers for population biology J. Trends in Ecology & Evolution Chicago University 29 Boivin T Bouvier J C Beslay D et al. Variability in diapause propensity within populations of a temperate insect species interactions between insecticide resistance genes and photoperiodism J. Biol J Linn Soc Crawford A M Littlepohn R P. The use of DNA marker in deciding conservation priorities in sheep and other livestock J. Anim Genet Resour Inf 櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍櫍 34 4 Farooq M Sulochana K N Pan X et al. Histone deacetylase 3 hdac3 is specifically required for liver development in zebrafish J. Dev Biol Zhang H Wei Q W H Du et al. Present status and risk for extinction of the Dabry s sturgeon Acipenser dabryanus in the Yangtze River watershed a concern for intensified rehabilitation needs J. J Appl Ichthyol Que Y F Xu D M Shao K et al. Identification and characterization of seventeen novel microsatellite markers for Dabry 's sturgeon Acipenser dabryanus J. J Genet dead end 5 Bruria F Elena O. Growth /differentiation factor - 11 an evolutionary conserved growth factor in vertebrates J. Dev Genes Evol C Ye H Yue H M Yang X G et al. Identification and sexually di- 6. morphic expression of vasa isoforms in Dabry's sturgeon Acipenser J dabryanus and functional analysis of vasa 3'- untranslated region 7. J. Cell Tissue Res J Gamperl A K Farrell A P. Cardiac plasticity in fishes environmental influences and intraspecific differences J. J Exp Biol 8 Du H Wei Q W Xie X et al. Improving swimming capacity of juvenile Dabry s sturgeon Acipenser dabryanus Dumeril 1869 in Pt current - enriched culture tanks J. J Appl Ichthyol Gallaugher P E Thorarensen H Kiessling A et al. Effects of high intensity exercise training on cardiovascular function oxygen uptake internal oxygen transport and osmotic balance in chinook salmon Oncorhynchus tshawytscha during critical speed swimming J. J Exp Biol

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2 : 111 ( Pinctada maxima), 13, 11,,,,,, 15, 25 cm, cm, 4 5 kg 1-2],,, 3-7] 24] 25-26],,, ] min, 1, 5 m 4 m, 20 m , 9-22],,, ( 12 2 2 0 1 6 4 South China Fisheries Science Vol. 12, No. 2 Apr., 2016 doi: 10. 3969/ j. issn. 2095-0780. 2016. 02. 016,,,, (,,, 510300) : ( Pinctada maxima),,,, 54. 55%, 0 0. 25 m - 2, 0 70. 31 gm - 2,