缢蛏ABC转运蛋白基因家族成员全基因组鉴定及表达模式分析

Genome-wide identification and expression pattern of ABC transporters in razor clam (Sinonovacula constricta)

中文关键词: 缢蛏  ABC转运蛋白  序列分析  表达模式

英文关键词: Sinonovacula constricta  ABC transporter  Sequence analysis  Gene expression pattern

中文摘要:

      腺苷三磷酸结合盒转运蛋白(ATP binding cassette transporter, ABC transporter)家族是最古老的膜蛋白家族之一，广泛存在于原核生物和真核生物体内，利用ATP水解释放的能量对氨基酸、脂质、抗生素等物质进行跨膜运输，参与生物体内多种生理活动。目前，对软体动物ABC转运蛋白家族的鉴定，仅在虾夷扇贝(Patinopecten yessoensis)、栉孔扇贝(Chlamys farreri)和长牡蛎(Crassostrea gigas) 3种双壳类中有系统研究，对缢蛏(Sinonovacula constricta) ABC转运蛋白家族的鉴定和表达模式分析尚未见报道。利用缢蛏基因组和转录组数据，运用本地及NCBI在线BLAST程序、FGENESH+、SMART、ExPASy、MEGA X、Mev 4.90和MapInspect等生物信息学分析工具，在全基因组水平系统地鉴定出52个缢蛏ABC转运蛋白，并对转运蛋白基因外显子数目、染色体定位等信息进行分析；通过系统进化分析，将缢蛏ABC转运蛋白家族分为8个亚家族，即ABCA~ABCH；通过比较分析不同物种ABC转运蛋白家族，推测基因串联复制事件对软体动物ABC转运蛋白家族成员数目的增加有影响。ABC转运蛋白基因在缢蛏不同发育时期和组织的表达分析显示，ABCC和ABCG亚家族多个基因在缢蛏稚贝时期表达量达到最高值，ABC转运蛋白基因在鳃、肝胰腺和性腺中表达种类较多。软体动物作为第二大动物类群，目前仅有3个物种的ABC转运蛋白得到了系统分类，缢蛏ABC转运蛋白基因家族的系统鉴定与表达模式分析为研究软体动物ABC转运蛋白基因的进化和缢蛏ABC转运蛋白功能提供了基础资料。

英文摘要:

      The razor clam (Sinonovacula constricta) is an economically important species and one of China´s four traditional mariculture mollusks. The ABC transporter family is one of the oldest membrane protein families, widespread across prokaryotes and eukaryotes. By utilizing the energy released by ATP hydrolysis, ABC transporters function to transfer amino acids, lipids, antibiotics, and many other substances across membranes, thereby participating in various physiological processes such as nutrition uptake, antigen presentation, drug excretion, and lipid homeostasis in living organisms. ABC transporters can be classified as full transporters, which contain two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs), a half-transporter (composed of one NBD and one TMD), and a non-transporter (composed of either two NBDs or two TMDs incapable of transporting). The NBD domain is responsible for binding and hydrolyzing ATP, whereas the TMD domain determines substrate specificity. NBD domain sequences are relatively more conserved. Several investigations of heavy metal pollution were conducted in mollusk culture areas, and it was found that the concentrations of some ions exceeded the limits. Previous studies have reported that the multixenobiotic resistance (MXR) mechanism in bivalves is mediated by ABC transporters from the ABCB, ABCC, and ABCG subfamilies, which are important for the cellular efflux of noxious metallic ions. Obtaining a greater understanding of ABC transporters may contribute to the development of a healthier and more scientific method of mollusk culture. Until now, the identification of the ABC transporter family in mollusks has only been systematically performed in three bivalves: Patinopecten yessoensis, Chlamys farreri, and Crassostrea gigas. The analysis and expression pattern of the ABC transporter family of the razor clam have not yet been reported. To systematically study ABC transporters and facilitate an understanding of the evolution and function of ABC transporters in razor clams and mollusks, depending on the genome and transcriptome data, 52 ABC transporter proteins were identified using the local and NCBI BLASTP, TBLASTN programs, and the ExPASy website. The SMART(simple modular architecture research tool) website and FGENESH+ were used to further predict the domain of ABC transporters. After applying the local BLASTN program to the published genome data of the razor clam, the MapInspect software and the CSDS (gene structure display server) website were used to generate graphical representation of the locations of ABC transporter genes on chromosomes and gene structures, respectively. Six vertebrates (Homo sapiens, Mus musculus, Xenopus laevis, Carassius auratus, Danio rerio, and Petromyzon marinus) and seven invertebrates (Diaphorina citri, Tetranychus urticae, Zeugodacus cucurbitae, Patinopecten yessoensis, Chlamys farreri, Crassostrea gigas, and Sinonovacula constricta) were chosen to compare and discuss the differences in subfamilies among species of various evolutionary status. Using the Mev 4.90 software, based on transcriptome data, the heat map of ABC transporter genes expression levels in eight tissues (gill, foot, adductor muscle, hepatopancreas, mantle, siphon, female gonad, and male gonad) and eight development stages (egg, cell, blastula, gastrula, trochophora, d-shaped larvae, umbo larvae, and spat) of the razor clam was completed. Based on sequence similarity, MEGA X software was used to divide the 52 ABC transporters into eight subfamilies, namely ABCA~ABCH, and phylogenetic trees of ABC transporters from four bivalves were drawn. The total of 52 ABC transporter genes was divided into 7 ABCA, 10 ABCB, 13 ABCC, 3 ABCD, 1 ABCE, 3 ABCF, 14 ABCG, and 1 ABCH. The ORF lengths of these genes ranged in size from 1 588 to 7 224 bp, the number of exons ranged from 8 to 44, and the deduced proteins were between 455 and 4 560 amino acids in length. Based on the composition of the protein domain, 52 ABC transporters could be divided into 24 full transporters, 24 half transporters, and four non-transporters. The ABCA subfamily consisted of 1 ABCA1, 2 ABCA2, 2 ABCA3, 1 ABCA5, and 1 ABCA12, all of which were full transporters. The ABCB subfamily consisted of four complete transporters, namely ABCB1a~ABCB1d, and six half transporters, namely ABCB6~ABCB9 and ABCB10a~ABCB10b. The ABCC subfamily comprised 7 ABCC1, 1 ABCC4, 3 ABCC5, 1 ABCC8, and 1 ABCC10, all of which were full transporters. The ABCD subfamily included ABCD2~ABCD4, all of which were half transporters. Non-transporters were observed in the ABCE and ABCF subfamilies, namely ABCE1 and ABCF1~ABCF3. The ABCG subfamily, consisting of 8 ABCG1, 1 ABCG2, 4 ABCG5, and 1 ABCG8, was the largest subfamily and its members were all half transporters. The ABCH subfamily contained only one member, ABCH1, which was a half-transporter. The comparison of ABC subfamilies between different species revealed that tandem duplication events might have resulted in an increase in the numbers of several ABC transporter genes during molluscan evolution and that some genes with functions related to immunity, such as ABCB1 and ABCC5, had multiple copies, indicating a positive influence on the environmental adaptation of mollusks. The analysis of the expression level of ABC transporter genes in different tissues and developmental stages of razor clam showed that the gill and hepatopancreas have relatively more expressed genes, which may be because of their detoxification function. The expression of ABCA and ABCG subfamily genes increased with razor clam development, and the expression levels of many ABCC and ABCG subfamily genes peaked in the spat stage. In general, several members of the ABCB subfamily, as well as all ABCE and ABCF subfamily genes, remained highly expressed in all eight tissues and all eight development stages. The ABC transporter gene family has only been investigated in three species of mollusks. Systematic identification and expression pattern analysis of ABC transporters in razor clams can promote our understanding of the evolution of ABC transporters in mollusks and provide an essential foundation for functional research on ABC transporters in mollusks, which may contribute to healthier mollusk cultur