单 位：
广东海洋大学水产学院;南方海洋科学与工程广东省实验室;广东省珍珠科技创新中心
关键词：
马氏珠母贝;PEPCK;低温耐受;SNP;基因克隆
摘 要：
磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase,PEPCK)是糖异生途径的关键酶,当动植物处于温度胁迫等不良环境条件时,它可通过催化草酰乙酸转化为磷酸烯醇式丙酮酸,继而生成葡萄糖以维持机体的血糖平衡,在抗逆过程中起着重要作用。本研究鉴定了马氏珠母贝(Pinctada fucata martensii)的PEPCK基因(Pm PEPCK),分析了低温胁迫下Pm PEPCK表达量的变化,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 PEPCK外显子区的SNP位点。序列分析表明,Pm PEPCK基因全长2557 bp,5'UTR和3'UTR分别为56 bp和446 bp,开放阅读框2055 bp,编码684个氨基酸。Pm PEPCK在所检测组织中均有表达,其中肝胰腺表达量最高(P<0.05)。低温胁迫后鳃组织中的时序表达结果显示,低温组(12℃、17℃) Pm PEPCK的表达量在6 h达到最高,显著高于22℃对照组(P<0.05),说明Pm PEPCK参与贝体的低温响应过程。对Pm PEPCK的外显子区SNP进行分析,共得到108个SNP,其中47个SNP位点的基因型频率在R和W群体间差异显著(P<0.05),仅在R中被检测到的基因型有11个,分别是位点g.60010420、g.60007411、g.60011365的基因型AG,位点g.60006265、g.60006166的基因型AA,位点g.60007389、g.60011387的基因型CT和g.60009449、g.60006028的基因型TT,位点g.60011341的基因型GG和g.60009464的基因型GT。结论认为经过3代选育后,R群体的Pm PEPCK基因受到了强烈的正选择,Pm PEPCK的这些SNPs及其基因型可能与选育系的低温耐受能力相关,并可能作为选择育种的候选标记。
译 名：
Sequence characteristics of PEPCK gene of Pinctada fucata martensii and its selection in low temperature-resistant line
作 者：
CHEN Kun;LAI Zhuoxin;LIU Ya;HAO Ruijuan;WANG Qingheng;Fisheries College, Guangdong Ocean University;Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang);Guangdong Science and Innovation Center for Pearl Culture;
关键词：
Pinctada fucata martensii;;PEPCK;;low temperature tolerance;;SNPs;;gene cloning
摘 要：
Phosphoenolpyruvate carboxylase is a key enzyme in the gluconeogenesis pathway. When animals and plants are under adverse environmental conditions such as temperature stress, they can convert oxaloacetic acid into phosphoenolpyruvate, generating glucose to maintain the blood glucose balance. This process plays an important role in stress resistance. In this study, PEPCK gene of Pinctada fucata martensii was identified, the changes of PmPEPCK expression under low temperature stress were analyzed, and single-nucleotide polymorphism(SNP) sites in the exon region of PmPEPCK in low temperature tolerant breeding line F3(R) and Beibu Gulf wild populations(W) of Pinctada fucata martensii were screened and compared. Sequence analysis showed that the PmPEPCK gene had a full length of 2557 bp; the 5'UTR and 3'UTR were 56 bp and 446 bp long, respectively, and the open reading frame was 2055 bp long and encoded 684 amino acids. PmPEPCK was expressed in the tested tissues, among which the hepatopancreas had the highest expression(P<0.05). The time-series expression results in gill tissues after low temperature stress showed that the expression of PmPEPCK in the low temperature group reached the highest at 12 ℃ and 17 ℃ at 6 h, which was significantly higher than that at 22 ℃noted in the control group(P<0.05), indicating that PmPEPCK participates in the low temperature response process of shellfish. A total of 108 SNPs were identified in the exon region of PmPEPCK, of which 47 SNPs showed significant differences between the R and W population(P<0.05). Among the 47 SNP loci, 11 genotypes only appeared in R, which were the AG genotype of loci g.60010420, g.60007411, and g.60011365; genotype AA of loci g.60006265 and g.60006166; genotype CT of loci g.60007389 and g.60011387; genotype TT of loci g.60009449 and g.60006028; genotype GG of locus g.60011341; and genotype GT of locus g.60009464. Thus, after 3 generations of selection, the PmPEPCK gene of the R population was strongly positively selected. These SNPs and their genotypes of PmPEPCK may be related to the low temperature tolerance of the breeding line and used as candidate markers for selective breeding.
