Progress in Fishery Sciences (Dec 2023)
Sequence Characteristics of Rab7 of Pinctada fucata martensii and Its Relationship with Low Temperature Tolerance
Abstract
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<PIC<0.5) in the R population. There were also no pairs of sites with complete linkage disequilibrium (D'=1, R2=1), one pair with strong linkage disequilibrium (0.8≤D'<1, 0.3≤R2<1), and eight pairs with weak linkage disequilibrium (D'<0.8, R2<0.3). In addition, evaluations of the W population revealed four low polymorphism loci (PIC<0.25) and three moderate polymorphism loci (0.25<PIC<0.5). Taken together, these results suggest that these loci are likely to be associated with improved low temperature resistance in P. f. martensii. Thus we conclude that Pm-Rab7 may play an important role in the adaptation of P. f. martensii to low temperatures, and that these findings may support further explorations into the adaptation of P. f. martensii to low temperatures.
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