MOLECULAR CHARACTERISTICS, GENOMIC STRUCTURE AND EXPRESSION PATTERNS OF DIVERSE BMP15 ALLELES IN POLYPLOID GIBEL CARP CLONE F
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摘要: 为探究骨形态发生蛋白15(Bone morphogenetic protein 15, bmp15)在银鲫(Carassius gibelio Bloch) F系卵巢发育过程中的表达特征, 研究采用cDNA末端快速扩增(RACE)方法首先从银鲫F系卵巢cDNA中克隆了2个bmp15部分同源基因(Homeologs), 将其命名为Cgbmp15a和Cgbmp15b, 它们各自具有3个不同等位基因。不同脊椎动物Bmp15蛋白序列多重比对和系统进化树均表明, 银鲫为异源六倍体, 在其进化历程中发生了两轮多倍化, 其中早期的异源多倍化整合了来自2个原始祖先的染色体组, 导致银鲫和鲫的四倍体共同原始祖先基因组中包含bmp15a和bmp15b; 随后第二轮同源多倍化最终导致银鲫存在6个bmp15等位基因。bmp15基因及其邻近基因同线性分析表明, 银鲫在形成异源多倍体后, 其基因组发生了复杂的变化, 包括bmp15邻近基因的丢失。Cgbmp15a和Cgbmp15b均主要在卵巢中表达, 在皮质泡期卵母细胞中表达量最高; 无论是在银鲫成体组织还是不同发育阶段的卵子中, Cgbmp15a的表达水平显著高于Cgbmp15b的表达水平。在孕酮激素DHP体外诱导银鲫F系GV1期卵母细胞6h后, Cgbmp15a开始上调表达, 诱导8h后达到最高表达水平, 随后下降; 而Cgbmp15b在诱导8h后表达水平有微弱上调, 随后下降。上述的结果表明, Cgbmp15a和Cgbmp15b在银鲫F系成体组织和不同发育和成熟阶段的卵子中, 均表现出偏性表达的特征, 暗示Cgbmp15a在银鲫的卵母细胞发育和成熟中起着主要作用。Abstract: Bone morphogenetic protein 15 (bmp15) plays key roles in regulating development and function maintenance of reproductive system in vertebrates. In this study, we first identified two divergent bmp15 homeologs from gibel carp (Carassius gibelio Bloch) clone F. Both Cgbmp15a and Cgbmp15b possess 3 different alleles, which were obtained from ovary cDNA by using rapid-amplification of cDNA ends (RACE). Multiple sequence alignment of Bmp15 protein in vertebrates and phylogenetic analysis of gibel carp, crucian carp, and zebrafish bmp15 genomic sequences indicated that two rounds of polyploidization had been occurred during allo-hexaploid gibel carp evolution. The early polyploidy produced the two divergent bmp15a and bmp15b, and the late polyploidy generated the 6 Cgbmp15 alleles of gibel carp. Synteny analysis of genomic regions around bmp15 gene in vertebrates suggested that the complex genomic changes might occur after the polyploidization in gibel carp, such as the loss of bmp15 adjacent genes. Cgbmp15a and Cgbmp15b were predominantly expressed in ovary with the highest level in cortical alveolus oocytes. The Cgbmp15a level is remarkably higher than Cgbmp15b in adult tissues and oocytes at different developmental stages (P<0.05). Moreover, Cgbmp15a increased at 6 hours and reached peak level at 8 hours after DHP induction in vitro, while Cgbmp15b slightly increased at 8 hours after DHP induction. These results indicate that Cgbmp15a play a major role in gibel carp oocyte development and maturation.
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Keywords:
- Gibel carp /
- Allo-hexaploid /
- Bmp15 /
- Oogenesis and oocyte maturation /
- Biased expression
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图 1 银鲫Cgbmp15a和Cgbmp15b的3个等位基因的基因组结构
方框表示外显子, 实线表示内含子; 方框/细线上方对应数字表示外显子/内含子大小(bp)
Figure 1. Schematic diagram for gene structure of gibel carp Cgbmp15as and Cgbmp15bs
Exons are indicated with boxes and intron with line. The number on the box/line indicates the size of exon/ intron (bp)
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图 2 银鲫Cgbmp15a和Cgbmp15b基因与其邻近基因保守的共线性关系(数据来自Ensemble数据库)
Figure 2. Schematic diagram for bmp15 and context genes of gibel carp Cgbmp15a and Cgbmp15b BACs and the bmp15 homologs of other species from Ensemble database
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图 3 ML法构建的银鲫、鲫和斑马鱼bmp15基因的系统进化树
分支上数值代表置信度, 设置1000次bootstrap进行评估
Figure 3. The phylogenetic tree of gibel carp, crucian carp, and zebrafish bmp15 based on ML method
The numbers on the branches indicate the hits supporting the branching pattern from 1000 bootstraps
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图 4 Cgbmp15a和Cgbmp15b mRNA在银鲫不同成体组织中的表达分析
以β-actin为内参; Mean±SD (n=3); *表示Cgbmp15a和Cgbmp15b在同一组织中表达水平具有显著性差异(P<0.05); 下同
Figure 4. Relative expression of Cgbmp15a and Cgbmp15b in different adult tissues
β-actin was used as control. Each bar represents mean ± standard deviation (SD, n=3). Asterisks (*) indicate significant differences (P<0.05) between Cgbmp15a and Cgbmp15b expression in same tissue. The same applies below
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图 5 不同发育阶段卵母细胞(A)以及DHP体外诱导GV1期卵母细胞成熟过程(B)中Cgbmp15a和Cgbmp15b的表达分析
Figure 5. Expression of Cgbmp15a and Cgbmp15b in oocytes during oogenesis (A) and GV1 oocytes induced by DHP in vitro (B)
表 1 本文中所用引物
Table 1 Primers used in this study
引物名称Primer 序列Sequence (5′－3′) 应用Usage Cgbmp15aF GGTCACGACTGTCTGAGA BAC克隆筛选 Cgbmp15aR GAGTCTGCAATCATGTCTTC Cgbmp15bF CTACCAGCAGTCACACCT Cgbmp15bR GAGTCTGCAATCATGTCTTC Cgbmp15a-5′-Outer TATACAGGATAGCGGTGGAT RACE Cgbmp15a-5′-Inner ACTGTAAGGCTGCTTCAAG Cgbmp15b-5′-Outer GGCGTCACCAATACAGAA Cgbmp15b-5′-Inner ACAAGTTATTCGGCTCCAA Cgbmp15a-3′-Outer GAGTCTGCAATCATGTCTTC Cgbmp15a-3′-Inner GCAGGATACGAGGACAATC Cgbmp15b-3′-Outer GAGTCTGCAATCATGTCTTC Cgbmp15b-3′-Inner CTCCATCATCAGCACACT UPM CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT NUP CTAATACGACTCACTATAGGGC Cgbmp15a QF GGGTCCAACACTGTAAGGC RT-qPCR Cgbmp15a QR CACCAATTTGTCCAACGAG Cgbmp15b QF TTATTCGGCTCCAACACTG Cgbmp15b QR TGTCCAACGAGAGGTTTTCC β-actin-QF AGCACGGTATTGTGACTAACTG β-actin-QR TCGAACATGATCTGTGTCATC 
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