Expression, Genetic Variation of IP3R3 Gene and Its Effect on Eggshell Quality in Sansui Duck (Anas platyrhyncha domestica)
TAN Guang-Hui1, 2, PING Li-Feng1, 2, XIONG Jian-Min1, 2, QIN Yuan-Yu1, 2, WU Lei1, 2, LI Jie-Zhang
1 College of Animal Science, Guizhou University, Guiyang 550025, China; 2 Key Laboratory of Genetics, Breeding and Reproduction of Plateau Mountain Animals, Ministry of education, Guiyang 550025, China
Abstract Inositol triphosphate receptor type Ⅲ(IP3R3) is mediator of second messenger-induced intracellular calcium release. It can combine with inositol triphosphate (IP3) to form calcium channel and regulate intracellular calcium release. In order to explore the genetic effects of IP3R3 gene on duck (Anas platyrhyncha domestica) eggshell quality and enrich the molecular genetic basis of important economic traits related to laying ducks. In this study, qRT-PCR was used to detect the tissue expression of IP3R3 gene in Guizhou Sansui ducks. According to the sequence of IP3R3 gene in GenBank, PCR amplification primers were designed, and the amplified products were recovered, purified and sequenced directly.. The SNP loci of 120 Sansui ducks were screened and identified by DNA Star software MegAlign program and sequencing peak map, and SPSS18.0 general linear model (GLM) was used to analyze the relationship between SNPs. The results showed that IP3R3 gene expression was detected in 11 tissues of ducks. The results showed that the mRNA of IP3R3 gene was detected in 11 tissues of ducks, among which the expression abundance of eggshell gland was the highest, and the relative expression quantity of lung, chest muscle and muscle stomach was the lowest. The expression level of IP3R3 gene in different tissues of duck was as follow: Eggshell glands>heart>spleen>glandular stomach>pancreas>kidney>liver>small intestine>lung>pectorals>muscular stomach. SNPs identification results showed that 4 SNPs were found in the IP3R3 gene, including g.21663 C>T and g.21699 G>A in exon 20, g.21646 G>C in intron 19, and g.21825 C>A in intron 20. Further analysis showed that exons g.21663 C>T and g.21699 G>A were synonymous mutation sites, which did not cause changes in tyrosine (TAC) and lysine (AAG). The results of genetic analysis of 4 SNP loci showed that all four SNP loci were moderately polymorphic and deviated from Hardy-Weinberg equilibrium (P<0.05) and results of association analysis showed that the influence of g.21646 on the egg shape index reached a significant level (P<0.05), in which the GG egg shape index was significantly higher than the CC genotype, and the effect of g.21663 on eggshell strength reached a significant level (P<0.05), among which the CT genotype eggshell strength was significantly higher than the TT and CC genotypes, and the other 2 SNP sites did not reach a significant level. Comprehensive correlation analysis and expression results showed that IP3R3 gene expression in the uterus of laying ducks was higher than that in other tissues. It was preliminarily speculated that the high expression of IP3R3 in the eggshell glands might be related to the regulation of eggshell quality, and the mutations of g.21663 and g.21646 loci had significant effects on duck eggshell quality, which could be used as candidate markers assisted by molecular markers for duck eggshell quality traits and provide scientific data for improving duck eggshell quality and poultry breeding.
[1] 黄晨轩, 岳巧娴, 郝二英, 等. 2018. 饲粮钙水平对蛋鸡产蛋后期生产性能、蛋品质和血钙相关指标动态变化的影响[J]. 中国家禽, 40(24): 25-29. (Huang C X, Yue Q X, Yue E Y, et al.2018. Effects of dietary calcium level on production performance, egg quality and serum biochemical parame[J]. China Poultry, 40(24): 25-29.) [2] 宋凌子. 2019.禽蛋钙化壳中有机基质组分及其与蛋壳性状相关性的研究[D]. 硕士学位论文, 浙江农林大学,导师: 刘长国, pp. 1-5. (Song L Z.2019. The organic matrix components in the poultry calcified shells and the correlations between the organic matrix and eggshell traits [D]. Thesis for M.S., Dissertation., Zhejiang A & F University. Suppervisor: Liu C G, pp. 1-5.) [3] 向光明, 刘秋月, 王翔宇, 等. 2019. PER1基因组织表达及多态性与绵羊季节性繁殖的相关性研究[J]. 农业生物技术学报, 27(07): 1215-1223. (Xiang G M, Liu Q Y, Wang X Y, et al.2019. Tissue expression and polymorphism of PER1 gene and their association with seasonal reproduction in sheep (Ovis aries)[J]. Journal of Agricultural Biotechnology, 27(07): 1215-1223.) [4] 杨宁主编. 2010. 家禽生产学[M]. 北京: 中国农业出版社, pp. 269-272. (Yang N. eds. 2010. Poultry production science [M]. China Agriculture Press, Beijing, China, pp. 269-272.) [5] 杨智青, 陈应江, 金崇富, 等. 2013, 防护林散养草鸡不同产蛋期蛋形、蛋壳指数比较[J].畜牧兽医杂志, 32(06): 12-14. (Yang Z Q, Cheng Y J, Jin C F, et al.2013.Comparison of egg shape and eggshell index in different laying stages of protective forest free-growing chickens[J]. Journal of Animal Husbandry and Veterinary Medicine, 32(06): 12-14. ) [6] Atala, Anthony.2018. Re: PTEN counteracts FBXL2 to promote IP3R3- and Ca2+-mediated apoptosis limiting tumour growth[J]. The Journal of Urology, 200(1): 31-32. [7] Bononi A, Giorgi C, Patergnani S, et al.2017. BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation[J]. Nature, 546(7659): 549-553. [8] Bultynck G.2016. Onco-IP3Rs feed cancerous cravings for mitochondrial Ca2+[J]. Trends in Biochemical Sciences, 41(5): 390-393. [9] Chamero P, Weiss J, Alonso, et al.2017. Type 3 inositol 1, 4, 5-trisphosphate receptor is dispensable for sensory activation of the mammalian vomeronasal organ[J]. Scientific Reports, 7: 10260. [10] Clapp T R, Stone L M, Margolskee R F, et al.2001. Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction[J]. BMC Neuroscience, 2(1): 6-10. [11] Enyedi P, Szabadkai G, Horváth A, et al.1994. Inositol 1, 4, 5-trisphosphate receptor subtypes in adrenal glomerulosa cells[J]. Endocrinology, 134(6): 2354-2359. [12] Filadi R, Leal N S, Schreiner B, et al.2018. TOM70 sustains cell bioenergetics by promoting IP3R3-mediated ER to mitochondria Ca2+ transfer[J]. Current Biology, 28(3): 369-382. [13] Franca A, Carlos M, Lima F, et al.2019. Effects of endotoxin on type 3 Inositol 1, 4, 5-trisphosphate receptor in human cholangiocytes[J]. Hepatology, 69(2): 817-830. [14] Fukuda N, Shirasu M, Sato C, et al.2008. Decreased olfactory mucus secretion and nasal abnormality in mike lacking type 2 and type 3 IP3 receptors[J]. European Journal of Neuroscience, 27(10): 2665-2675. [15] Hsiao C T, Liu Y T, Liao Y C, et al.2018. Mutational analysis of ITPR1 in a Taiwanese cohort with cerebellar ataxias[J]. PLOS ONE, 13(2): e0187503. [16] Huang Y C, Lin Y J, Chang J S, et al.2010. Single nucleotide polymorphism rs2229634 in the itpr3 gene is associated with the risk of developing coronary artery aneurysm in children with kawasaki disease[J]. International Journal of Immunogenetics, 37(6): 439-443. [17] Iommelli F, De Rosa V, Terlizzi C, et al.2018. Inositol trisphosphate receptor type 3-mediated enhancement of EGFR and MET co-targeting efficacy in non-small cell lung cancer detected by 18F-fluorothymidine[J]. Clinical Cancer Research, 24(13):3126-3136. [18] Jia C, Hegg C C.2015. Effect of IP3R3 and NPY on age-related declines in olfactory stem cell proliferation[J]. Neurobiol Aging, 36(2): 1045-1056. [19] Khan S, Wu S B, Roberts J.2019.RNA-sequencing analysis of shell gland shows differences in gene expression profile at two time-points of eggshell formation in laying chickens[J]. BMC Genomics, 20(1): 89-100. [20] Kim S, Ahn T, Park C.2005. The Pro335 - Leu polymorphism of type 3 inositol 1, 4, 5-trisphosphate receptor found in mouse inbred lines results in functional change[J]. Journal of Biological Chemistry, 280(28): 26024-26031. [21] Lee M G, Xu X, Zeng W, et al.1997. Polarized expression of Ca2+ channels in pancreatic and salivary gland cells: Correlation with initlation and propagation of Ca2+ waves[J]. Journal of Biological Chemistry, 272(25): 15765-15770. [22] Mayo O.2008. A century of Hardy-Weinberg equilibrium[J]. Twin Research and Human Genetics, 11(3): 249-256. [23] Okeke E, Parker T, Dingsdale H, et al.2016. Epithelial-mesenchymal transition, IP3 receptors and ER-PM junctions: Translocation of Ca2+ signaling complexes and regulation of migration[J]. Biochemical Journal, 473(6): 757-767. [24] Shibao K, Fiedler M J, Nagata J, et al.2010. The type Ⅲ inositol 1, 4, 5-trisphosphate receptor is associated with aggressiveness of colorectal carcinoma[J]. Cell Calcium, 48(6): 315-323. [25] Siefjediers A, Hardt M, Prinz G, et al.2007. Characterization of inositol 1, 4, 5-trisphosphate (IP3) receptor subtypes at rat colonic epithelium[J]. Cell Calcium, 41(4): 303-315. [26] Tang H, Wang H, Lin Q, et al.2017. Loss of IP 3 receptor-mediated Ca2+ release in mouse B cells results in abnormal B cell development and function[J]. The Journal of Immunology, 199(2): 570-580. [27] Wei W, Huang W, Yue J.2016. Requirement of IP3 receptors 3 (IP3R3) in nitric oxide induced cardiomyocyte differentiation of mouse embryonic stem cells[J]. Experimental Cell Research, 346(1): 9-16. [28] Yang Y C, Chang T Y, Chen T C, et al.2017. ITPR3 gene haplotype is associated with cervical squamous cell carcinoma risk in Taiwanese women[J]. Oncotarget, 8(6): 10085-10090. [29] Zhang Z, Liu M, Guan Z, et al.2016. Disbalance of calcium regulation-related genes in broiler hearts induced by selenium deficiency[J]. Avian Pathology, 46(3): 265-271.
