ClONE AND EXPRESSION ANALYSIS OF ORG GENE IN YANGTZE STURGEON (ACIPENSER DABRYANUS)
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摘要: 为了研究卵子发生相关基因org基因在长江鲟(Acipenser dabryanus)卵子发生过程的作用, 克隆得到长江鲟org基因(命名为Adorg)的全长cDNA序列, 该序列为1031 bp, 编码233个氨基酸。氨基酸序列比对分析发现, 长江鲟AdOrg与斑马鱼(Danio rerio)ZOrg蛋白序列的一致性最高, 为49.5 %。荧光定量PCR研究发现, 长江鲟Adorg mRNA特异地表达于性腺, 其在卵巢大量表达, 在精巢微量表达, 而在其他组织(肝、肠、脾、肾、心、肌肉、鳃、垂体和下丘脑)均未检测到其表达; 胚胎发育过程的动态表达分析表明, Adorg为母源表达, 在原肠胚之前均具有较高的表达水平, 随后其表达量急剧下降; 进一步研究其在卵子发生的表达模式, 发现Adorg基因在未分化性腺中的表达量极低, 随着卵母细胞的生长发育, 其mRNA表达水平急剧上升, 且维持在非常高的水平, 在Ⅱ期卵巢中的表达量最高。性腺切片RNA原位杂交实验结果表明, Adorg特异地在生殖细胞表达; 在卵巢中, Adorg在卵原细胞的信号较弱, 但在初级卵母细胞中, 其信号急剧增强且分布在卵母细胞的胞质, 且随着初级卵母细胞的生长发育, 其信号也逐渐增强; 在精巢中, 发现Adorg mRNA在A型和B型精原细胞中表达, 而在初级和次级精母细胞中未发现阳性信号。上述结果表明, Adorg基因可能在长江鲟性别分化和卵子发生过程中都起重要作用, 有待通过基因敲降或敲除技术来进一步阐明其功能。Abstract: Yangtze sturgeon, Acipenser dabryanus, is a freshwater fish that mainly distributed in the upper reaches of Yangtze River and its tributaries. Male and female Yangtze sturgeons in the wild reach sexual maturity at 4—6 and 6—8 years, respectively. Currently, it is a critically endangered species due to overfishing, habitat degradation and pollution. In order to protect and restore this species, many efforts have been made, such as building nature reserves and fishery stock enhancement and releasing. However, the wild population of Yangtze sturgeon has sharply decreased, and it is hardly observed in the Yangtze River now. Previous studies, such as diet supplemented with exogenous hormone and juveniles injected with peptide, were performed to short its sexual maturation time, but no effect has been observed. The oogenesis-related gene, org, plays an important role in the growth and development of oocytes in teleost fish. In order to reveal its function during the oogenesis of Yangtze sturgeon, a full-length cDNA of an org homologue was isolated (designated as Adorg), which was 1031 bp, encoding 233 amino acids. Multiple sequence alignments showed that AdOrg shared the highest sequence identity (49.5%) with zebrafish. By quantitative real-time PCR analysis, Adorg mRNA was specifically transcribed in the gonad, abundant in the ovary and weak in the testis. Transcription of Adorg was not detected in other somatic tissues including liver, intestine, spleen, kidney, heart, muscle, gill, pituitary and hypothalamus. During embryogenesis, Adorg was proved to be maternally transcribed, maintaining a high level before the gastrula stage, and then declining dramatically in later developmental stages. The transcription of Adorg was very limited in undifferentiated gonads, but it sharply increased in the following process of oogenesis, with its highest expression in stage Ⅱ oocytes. In situ hybridization of gonad indicated that the signal of Adorg mRNA was specifically located in the germ cells. In the ovary, Adorg signal was weak in the oogonia, and it increased rapidly in the cytoplasm of primary oocytes, and became stronger with the development of oocytes. In the testes, the expression of Adorg was restricted to type A and B spermatogonia, and was barely detectable in spermatocytes. These findings suggested that Adorg gene might play vital roles not only in oogenesis, but also in development and differentiation of germ cells. Knock-down or knock-out of this gene is necessary for exploring its specific function. These results paved the way for understanding the function of Adorg gene in Yangtze sturgeon during oogenesis.
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Keywords:
- Acipenser dabryanus /
- org gene /
- mRNA expression analysis /
- Oogenesis
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图 1 长江鲟AdOrg与其他鱼类Org蛋白序列比对
Figure 1. Protein sequence alignment of Yangtze sturgeon AdOrg with that of other fish homologues
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图 2 长江鲟Adorg基因在成鱼不同组织的表达水平
Figure 2. The expression of Adorg gene in different adult tissues of Yangtze sturgeon
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图 3 长江鲟不同发育时期性腺的组织学特征
A. 未分化的性腺; B. Ⅰ期卵巢; C. Ⅱ期卵巢; D. Ⅲ期卵巢; E. Ⅳ期卵巢; F. Ⅱ期精巢; G. 性腺; Og. 卵原细胞; SG. 精原细胞; PSP, 初级精母细胞
Figure 3. Histological characteristics of different stages of Yangtze sturgeon gonads
A. undifferentiated gonad; B. ovary at the stage I; C. ovary at the stage Ⅱ; D. ovary at the stage Ⅲ; E. ovary at the stage Ⅳ; F. testis at the stage Ⅱ; G. gonad; Og. oogonia; SG. spermatogonia; PSP. primary spermatocytes
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图 4 长江鲟Adorg基因在未受精卵和不同发育时期胚胎的表达量
Figure 4. Expression levels of Adorg in unfertilized eggs and different development stages of Yangtze sturgeon embryos
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图 5 长江鲟Adorg基因在卵子发生过程中的表达量变化
Figure 5. Expression variation of Adorg gene during oogenesis
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图 6 Adorg基因在长江鲟卵巢和精巢的细胞定位
A. Adorg mRNA在卵巢的细胞定位; B. Adorg mRNA在精巢的细胞定位; C. 正义探针在卵巢中没有信号; D. 正义探针在精巢中没有信号; Og. 卵原细胞; SG. 精原细胞; PSP. 初级精母细胞; SSP. 次级精母细胞
Figure 6. Cellular localization of Adorg gene in the ovary and testis of Yangtze sturgeon
A. localization of Adorg mRNA in the ovary; B. localization of Adorg mRNA in the testis; C. signal of sense probe of Adorg mRNA in the ovary; D. signal of sense probe of Adorg mRNA in the testis. Og. oogonia; SG. spermatogonia; PSP. primary spermatocytes; SSP. secondary spermatocytes
表 1 本研究所用引物
Table 1 Primers used in this study
引物名称Primer 序列
Sequence (5′—3′)用途
UsageAdorg-3gsp1 GTGACAGCGAGGAGAACAGGACTC 3′RACE-PCR Adorg-3gsp2 ACTCAGAGCAGCAATCGTGATCC 3′RACE-PCR Adorg-5gsp1 TCGCTGCTCGGCTCGTAAGTGGGA 5′RACE-PCR Adorg-5gsp2 GGACAGGGTAGCCGAGCGCACTCT 5′RACE-PCR UPM CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAG和CTAATACGACTCACTATAGGGC RACE通用引物 NUP AAGCAGTGGTATCAACGCAGAGT RACE通用引物 Adorg-qF CTGGGAAGGGTAACAAGCGTAAAC 荧光定量PCR Adorg-qR CCTCTTCTGCCACCTCCTCTATG 荧光定量PCR Adorg-anti-F TGAGAAGCCGCCAGTTGTTG 原位杂交反义探针 Adorg-anti-R TAATACGACTCACTATAGGGCTGAGGCGTAATCTGTAAAACTTG 原位杂交反义探针 Adorg-sense-F TAATACGACTCACTATAGGGTGAGAAGCCGCCAGTTGTTG 原位杂交正义探针 Adorg-sense-R CTGAGGCGTAATCTGTAAAACTTG 原位杂交正义探针 
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