中华绒螯蟹Kcna基因功能及其与生长性状的关联分析

为探究中华绒螯蟹Kcna基因的结构、表达模式及生长发育中的分子功能，本研究克隆了中华绒螯蟹Kcna（命名为Es-Kcna）基因的全长，并开展了生物信息学分析和时空表达模式研究；观察了干扰Kcna基因后中华绒螯蟹生长表型性状的变化；筛选了Es-Kcna基因的SNP标记并与种群地理分布、生长性状进行了关联分析。结果显示：Kcna基因位于中华绒螯蟹第46号染色体上，全长945 304 bp，含有9个外显子；其中，cDNA全长2 080 bp，开放阅读框1 584 bp，编码527个氨基酸；原子总量为8 433，分子结构式为C2719H4210N702O787S15，预测蛋白等电点（pI）为5.23，相对分子量为59.81 KDa。系统进化树分析显示Es-Kcna基因与三疣梭子蟹Kcna基因的亲缘关系最近。荧光定量PCR结果显示Es-Kcna基因在蜕壳前期、蜕壳间期和蜕壳后期的肌肉、心脏、肠道等六种组织中均有表达，其中以肌肉组织中的表达丰度最高（P< 0.05）。与对照组相比，干扰Kcna基因的表达后，实验组蟹的体重、蜕壳增重率和第二步足长度显著降低（P< 0.05）；肌肉组织切片结果显示实验组步足肌肉肌纤维直径小于对照组（P> 0.05）。此外，在Es-Kcna基因的第8外显子上鉴定出一个SNP位点（A1 461G），该位点在辽河野生群体显著富集GG基因型，而在长江野生群体富集AA基因型。生长性状的关联分析表明，具有AA型个体的步足长度显著长于GG型个体（P< 0.05）。本研究为Kcna基因调控中华绒螯蟹生长的分子功能和遗传育种研究提供了研究基础，并为区分长江、辽河野生中华绒螯蟹提供重要参考。

Molecular function of Kcna gene and its association with growth traits in Eriocheir sinensis

In order to investigate the gene structure, expression pattern, and molecular function of Kcna gene in Eriocheir sinensis, this study cloned the full-length Kcna gene of E.sinensis (named Es-Kcna), and conducted bioinformatics analysis and spatio-temporal expression pattern research; observed the changes in growth phenotype characteristics of E.sinensis after RNA interfering with Es-Kcna gene; and screened the SNP molecular markers of Es-Kcna gene and conducted association analysis with population geographic distribution and growth characteristics. The results showed that Kcna gene was located on chromosome 46 of E.sinensis, with a length of 945,304 bp and nine exons. The full-length cDNA was 2 080 bp, the open reading frame was 1 584 bp, and encoded 527 amino acids, the total number of atoms is 8 433, the molecular structure formula is C2719H4210N702O787S15, the predicted isoelectric point (pI) is 5.23, and the relative molecular weight is 59.81 KDa. Phylogenetic tree analysis showed that Es-Kcna gene was most closely related to the Kcna gene of Portunus trituberculatus, and the crustaceans clustered into a separate clade. Fluorescence quantitative PCR (qRT-PCR) results showed that Es-Kcna gene was expressed in six tissues, including muscle, heart, intestine, eyestalk, gill and hepatopancreas at pre-molting, inter-molting and post-molting stages, with the highest expression abundance identified in muscle tissue (P < 0.05). Compared with the control group, the body weight, molting weight gain rate and second walking leg length of the experimental group crabs were significantly decreased after RNA interfering of the Kcna gene (P < 0.05). Muscle tissue slice results showed that the diameter of muscle fibers in the walking legs of the experimental group was smaller than that of the control group (P > 0.05). In addition, a SNP (A1,461G) was identified in the exon 8 of Es-Kcna gene. The association analysis of population distribution showed that this SNP was significantly enriched in the GG genotype in the wild population of Liaohe River, while the AA genotype was enriched in the wild population of Yangtze River. The association analysis of growth traits showed that the second walking leg length of individuals with the AA genotype was significantly longer than that of individuals with the GG genotype (P < 0.05). The present study provided insight into the molecular function on Kcna gene in regulating growth and potential utilization in genetic breeding of E.sinensis, and provided an important reference for distinguishing wild E. sinensis from the Yangtze and Liaohe rivers.