三疣梭子蟹氧化磷酸化代谢在家系近交中的变化

摘要

为探究近交对三疣梭子蟹(Portunus trituberculatus)氧化磷酸化代谢的影响,首先克隆了三疣梭子蟹线粒体呼吸链4个复合体关键亚基基因的cDNA全长,分别命名为ptNdufv2、ptSDHC、ptCytochrome c1与ptCOX6B.ptNdufv2基因cDNA全长1005 bp,编码234个氨基酸,该蛋白是复合体Ⅰ的核心亚基Ndufv2;ptSDHC基因全长915 bp,编码由179个氨基酸组成的复合体Ⅱ关键亚基SDHC;ptCytochrome c1基因全长2371 bp,编码由313个氨基酸组成的亚基Cyt c1;ptCOX6B基因全长1171 bp,编码由105个氨基酸组成的COX6B亚基.生物信息学分析显示,这些复合体亚基基因进化上比较保守.酶活检测及RT-PCR结果表明,随着近交系数的增加,三疣梭子蟹肝胰腺中复合体Ⅰ、复合体Ⅲ活力分别从F4、F2开始呈现显著下降趋势(P<0.05);F10复合体Ⅳ酶活力显著低于其余各代(P<0.05);4个复合体基因表达量均显著下降,且各代表达量显著低于F0(P<0.05).在心脏中,复合体Ⅰ、Ⅲ、Ⅳ活力分别从F2、F2、F6开始显著下降(P<0.05);ptNdufv2与ptCytochrome c1基因表达量分别从F2、F4开始显著下降(P<0.05),这一结果证实近交衰退已出现在三疣梭子蟹氧化磷酸化代谢通路.%The swimming crab Portunus trituberculatus (Crustacea: Decapoda: Brachyura) is a dominant species of portunid crab fisheries and an important economic species worldwide. The key performance traits of crustaceans and other aquaculture species are often depressed by inbreeding, especially if inbreeding effects accumulate too rapidly. Although previous studies have investigated the effects of inbreeding on the morphology, hatchability, and survival of crustaceans, little evidence is available that demonstrates that inbreeding affects crustacean physiology. Therefore, in order to investigate the effects of inbreeding on the oxidative phosphorylation of P. trituberculatus, cDNAs of the key subunit genes of the four mitochondrial respiratory chain complexes were cloned, sequenced using rapid amplification of cDNA ends (RACE), and then analyzed using bioinformatics technology. The full-length cDNA sequence of the Complex Ⅰ core subunit genes was 1005 bp in length and contained a 705-bp open reading frame (ORF) that encoded a 234-amino acid polypeptide (GenBank: KY682717). The full-length cDNA sequence of Complex Ⅱ was 915 bp in length and contained a 540-bp ORF that encoded a 179-amino acid polypeptide (GenBank: KY406169). The full-length cDNA sequence of the Complex Ⅲ subunit (Cytc1) gene was 2371 bp in length and contained a 942-bp ORF that encoded a 313-amino acid polypeptide (GenBank: KY406171), and the full-length cDNA for the key subunit of Complex IV was 1171 bp in length and encoded 105 amino acids (GenBank: KY406170). In addition, homology and phylogenetic analyses revealed that the amino acid sequences of the four complexes were highly similar to those of closely related species and had higher conservation in evo-lution, and could be used as a reference for other marine organisms. The activities and mRNA expression of the four complexes in the hepatopancreas and heart mitochondria of P. trituberculatus were investigated. Results show that inbreeding reduced the activity of all four complexes and their respective subunit gene in the hepatopancreas (P<0.05). Besides, Complex I, III and IV activities and their subunit genes in heart were declined by inbreeding (P<0.05). Furthermore, the elevated activity and expression of Complex II in the heart may indicate that the ability to oxidize succinic acid and the level of aerobic metabolism in the crabs rose by breeding might because the dominant homozygous genes were accumulated during the family-based selective breeding programs. Therefore, it is clear that inbreeding gradually reduces the oxidative phosphorylation pathway and provides a reference for family-based selective breeding programs for P. trituberculatus.