| 宋欣霖,杨佳雯,韩书雅,赖卓欣,王庆恒,邓岳文.马氏珠母贝Rab7基因序列特征及其与耐低温性状的关系.渔业科学进展,2023,44(6):155-165 |
| 马氏珠母贝Rab7基因序列特征及其与耐低温性状的关系 |
| Sequence characteristics of Rab7 of Pinctada fucata martensii and its relationship with low temperature tolerance |
| 投稿时间：2022-07-13 修订日期：2022-08-07 |
| DOI：10.19663/j.issn2095-9869.20220713001 |
| 中文关键词: 马氏珠母贝 Pm-Rab7 基因克隆 温度胁迫 SNP |
| 英文关键词: Pinctada fucata martensii Pm-Rab7 Gene cloning Temperature stress SNP |
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| 中文摘要: |
| Rab蛋白是膜泡运输过程中的重要调节因子，在囊泡运输和水产动物的免疫应答中行使重要功能。本研究鉴定了马氏珠母贝(Pinctada fucata martensii)的Rab7基因(Pm-Rab7)，分析了Pm-Rab7在6个组织中及在温度胁迫(低温组17 ℃、对照组22 ℃、高温组32 ℃)下的表达模式，筛选和比较分析了马氏珠母贝耐低温选育系(low temperature resistant line, R) F3和北部湾野生群体(Beibu Gulf wild population, W)的Pm-Rab7外显子区的SNP位点。结果显示，Pm-Rab7序列全长为1 153 bp，5′端、3′端和开放阅读框(ORF)序列长度分别为30、505和618 bp，共编码205个氨基酸；Pm-Rab7具有一个RAB保守结构域，并与太平洋牡蛎(Crassostrea gigas)相似度最高(92.79%)。系统进化树结果显示，Pm-Rab7与太平洋牡蛎等软体动物聚为一支。Pm-Rab7在所检测的组织都有表达，其中在性腺和鳃中的表达显著高于外套膜和肝胰腺等组织(P<0.05)。Pm-Rab7温度胁迫后时序表达分析发现，在17 ℃低温组，Pm-Rab7的表达量呈先上升再下降的趋势，在6 h~3 d内均显著高于22 ℃对照组(P<0.05)，在1 d时达到峰值；在32 ℃高温组，Pm-Rab7表达量总体稳定，仅12 h时显著高于对照组(P<0.05)，其他时间点与对照组无显著差异(P>0.05)；说明该基因的表达与马氏珠母贝对低温胁迫的响应密切相关。Pm-Rab7基因的外显子区域在马氏珠母贝耐低温选育系和北部湾野生群体中共检测到7个SNP位点，其中3个位点的基因型频率在2个群体间具有显著性差异，说明这些位点可能与其耐低温性状相关。结果表明，Pm-Rab7可能在马氏珠母贝耐低温适应过程中发挥重要作用，研究结果可为深入探讨马氏珠母贝对温度变化的环境适应性研究提供参考资料。 |
| 英文摘要: |
| The Ras-related proteins in brain (Rab) subfamily is the largest group of small molecule guanine nucleotide-binding regulatory proteins (GTP-binding proteins). These proteins are widely distributed across various eukaryotes and consist of approximately 200 amino acids, producing a molecular weight of approximately 20~25 kDa. Rab is known to be an important regulator in the transport of membrane vesicles and is embedded in almost all membrane-related proteins. Rab is primarily regulated through GTP binding and hydrolysis within the organelles and is known to perform several important functions across various stages of vesicle transport. Current research on Rab proteins produced by aquatic animals has revealed that the Rab gene plays an important role in immune response. The Pearl oyster, Pinctada fucata martensii, is the most common species used in the cultivation of seawater pearls, making them economically important. These oysters are primarily distributed along the coastal areas of Guangdong, Guangxi, and Hainan provinces in China. However, they display weak tolerance to low temperatures, which severely limits their cultivation area and the overall production of seawater pearls. Given the desire to expand their cultivation northward, we designed this study to help create low-temperature resistant P. f. martensii breeding lines F3 (R). We used genome resequencing technology to compare and analyze the R and Beibu Gulf wildtype populations (W), and identified a group of candidate genes, including the Rab gene, that were strongly positively selected during the breeding process. In addition, the full-length sequence of Rab7 of P. f. martensii (Pm-Rab7) was cloned using RACE technology, and the expression levels of Pm-Rab7 in adductor muscle, gill, gonad, hepatopancreas, foot, and mantle, and their expression patterns under temperature stress (17 ℃ in the low temperature group, 22 ℃ in the control group, and 32 ℃ in the high temperature group) were detected using bioinformatics analysis. Furthermore, single nucleotide polymorphisms (SNPs) in the exon regions of Pm-Rab7 in the R and W were screened, and this information was used to determine the genetic polymorphism, haplotype, and frequency rates for each mutation. Sequence analysis also revealed that the Pm-Rab7 gene displayed a full length of 1 153 bp, with the 5' and 3' UTR adding 30 bp and 505 bp, respectively. This gene was also shown to encode a single open reading frame (ORF) of 618 bp and 205 amino acids, producing a theoretical protein of 23.04 kDa with the isoelectric point at 5.40. This was later confirmed using the cloned Pm-Rab7 gene, which produced 5 conserved amino acid sequences, including a RAB conserved domain. Similarity evaluations revealed a high degree of overlap with C. gigas (92.79%), whereas phylogenetic tree construction revealed that the Rab7 gene produced a tree with three branches, one each for protostomes, echinoderms, and vertebrates. Closer inspection of the protostomes branch revealed that Pm-Rab7 first clusters with other mollusks, and then with arthropods to produce a large clade, suggesting that this protein is relatively well conserved during the evolutionary process. Pm-Rab7 was also shown to be expressed in all the tested tissues, with the most Pm-Rab7 expression recorded in the gonad and gill (P<0.05). Time-course results from the gill tissues of temperature stressed samples revealed that Pm-Rab7 expression first increased and then decreased in response to low temperature (17 ℃) exposure, with all time points showing a significant increase in Pm-Rab7 expression when compared to that in the 22 ℃ control from 6 h to 3 d (P<0.05). In addition, we noted an expression peak at 1 d. The expression of Pm-Rab7 was generally stable in response to growth at 32 ℃ (high temperature group), but was also significantly increased when compared to that in the control group following 12 h of exposure (P<0.05). This suggests that Pm-Rab7 is most likely linked to the low temperature response process in these shellfish. We also identified a total of seven SNPs in the exon region of Pm-Rab7, three (g.112712470, g.112712503, and g.11271477) of which demonstrated significant differences in occurrence rate between the R and W populations (P<0.05). Genetic evaluations of all seven SNPs revealed that three could be classified as low polymorphism loci (PIC<0.25) and four could be classified as moderate polymorphism loci (0.25 |
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