番鸭GDF9基因克隆、生物信息学及组织表达分析

doi:10.16431/j.cnki.1671-7236.2022.03.003

摘要/Abstract

摘要： 【目的】对番鸭（Cairina moschata）生长分化因子9（growth differentiation factor 9，GDF9）基因进行克隆及结构和功能的预测，并检测其在就巢期和产蛋期番鸭各组织中的表达差异。【方法】以番鸭卵巢组织cDNA为模板，利用快速扩增cDNA末端（rapid-amplification of cDNA ends，RACE）技术对GDF9基因进行扩增和克隆，利用生物信息学软件对GDF9基因的编码序列进行相似性分析和进化树构建，分析其基本理化性质和编码氨基酸序列的结构特征。利用实时荧光定量PCR技术检测就巢期番鸭和产蛋期番鸭各组织中GDF9基因的相对表达量。【结果】 GDF9基因CDS区全长1 380 bp，编码459个氨基酸；番鸭GDF9基因序列与GenBank已公布的凤头潜鸭、绿头鸭、天鹅、棕硬尾鸭、浙东白鹅、企鹅、鸡、牛、绵羊、猪和小鼠对应序列的相似性依次为98.8%、97.5%、96.4%、95.8%、94.4%、90.2%、87.5%、64.5%、64.2%、63.9%和60.6%。系统进化树结果显示，番鸭与凤头潜鸭和绿头鸭的亲缘关系较近，与小鼠的亲缘关系较远。GDF9蛋白分子质量为52.81 ku，是一种不稳定的碱性亲水膜外蛋白，有1个信号肽，含有45个磷酸化位点，8个O-糖基化位点，3个N-糖基化位点，存在1个转化生长因子-β（TGF-β）结构域；蛋白质的二级结构由无规则卷曲、α-螺旋、T-转角和β-折叠构成，所占比例分别为58.61%、22.22%、16.56%和2.61%。GDF9蛋白三级结构预测结果与二级结构一致。GDF9蛋白可能与BMP15、BMPR1B、BMPR2、TGFBR1、ACVR1B、POF1B、ZP2、ZAR1和MOS蛋白存在相互作用。实时荧光定量PCR结果显示，就巢期和产蛋期番鸭各组织中均有GDF9基因的表达，其中两个时期卵巢中的表达量均最高，且均极显著高于同一时期其他组织基因表达量（P<0.01）。同一组织不同时期相比，就巢期番鸭GDF9基因在脾脏、心脏和肾脏的表达量显著或极显著低于产蛋期（P<0.05；P<0.01），在卵巢中的表达量极显著高于产蛋期（P<0.01），其他组织中的表达量在两个时期间差异均不显著（P>0.05）。【结论】本研究成功克隆番鸭GDF9基因，GDF9基因在番鸭各组织中均有表达，GDF9基因为番鸭产蛋期和就巢期卵巢、心脏、肾脏和脾脏的差异表达基因。该研究为进一步探索番鸭GDF9基因的功能提供了理论依据，也为研究GDF9基因在卵巢发育中的生物学功能及其分子机制奠定基础。

关键词: 番鸭; GDF9基因; 卵巢; 生物信息学分析

Abstract: 【Objective】 The aim of this study was to clone and prediect its structure and function of growth differentiation factor 9 (GDF9) gene of Muscovy duck (Cairina moschata), and to detect its expression differences in Muscovy duck tissues at the nest stage and laying stage.【Method】 The ovary tissue cDNA in Muscovy duck was used as a template, the GDF9 gene was amplified and cloned by rapid-amplification of cDNA ends (RACE).Using biological information software, the similarity of the coding sequence of GDF9 gene was analyzed and evolutionary tree was constructed, and its basic physical and chemical properties and the structural characteristics of the coding amino acid sequence were analyzed.Real-time quantitative PCR was used to detect the expression of GDF9 gene in Muscovy duck of broody period and laying period.【Result】 The total CDS region sequence of GDF9 gene was 1 380 bp, which edcoded 459 amino acids.The similarity of GDF9 gene sequence of Muscovy duck with that of Aythya fuligula, Anas platyrhynchos, Cygnus atratus, Oxyura jamaicensis, Anser cygnodies domesticus, Aptenodytes forsteri, Gallus gallus, Bos taurus, Ovis aries, Sus scrofa and Mus musculus published in GenBank was 98.8%, 97.5%, 96.4%, 95.8%, 94.4%, 90.2%, 87.5%, 64.5%, 64.2%, 63.9% and 60.6%, respectively.The phylogenetic tree showed that Muscovy duck was closely related to Aythya fuligula and Anas platyrhynchos, but far from Mus musculus.The molecular weight of GDF9 protein was 52.81 ku.It was an unstable alkaline hydrophilic membrane protein.It had one signal peptide, 45 phosphorylation sites, 8 O-glycosylation sites, 4 N-glycosylation sites and a transforming growth factor-β (TGF-β) domain.The secondary structure of protein consists of random coil, alpha helix, T-turn and beta sheet composition, accounting for 58.61%, 22.22%, 16.56% and 2.61% respectively.The predicted results of the tertiary structure of GDF9 protein were consistent with the secondary structure.GDF9 protein might interact with BMP15, BMPR1B, BMPR2, TGFBR1, ACVR1B, POF1B, ZP2, ZAR1 and MOS proteins.The results of Real-time quantitative PCR showed that GDF9 gene was expressed in all tissues of Muscovy ducks at nesting and laying stages, the expression of genes in ovary were the highest in two stages, and they were extremely significantly higher than that in other tissues at the same time (P<0.01).Compared with different periods of the same tissue, the expression of GDF9 gene in spleen, heart and kidney in nesting Muscovy duck was significantly or extremely significantly lower than that in laying period (P<0.05 or P<0.01), and the expression in ovary was extremely significantly higher than that in laying period (P<0.01).There was no significant difference in the expression of GDF9 gene in other tissues between the two periods (P>0.05).【Conclusion】 In this study, the GDF9 gene of Muscovy duck was successfully cloned.GDF9 gene was expressed in all tissues of Muscovy duck.GDF9 gene was due to the differentially expressed genes in ovary, heart, kidney and spleen of Muscovy duck during egg laying and nesting.This study provided a theoretical basis for further exploring the function of GDF9 gene in Muscovy duck, and also laid a foundation for studying the biological function and molecular mechanism of GDF9 gene in ovarian development.

Key words: Muscovy ducks; GDF9 gene; ovary; bioinformatics analysis

中图分类号:

石雨竹, 罗儒唐, 陈超, 吴旭, 李昂. 番鸭GDF9基因克隆、生物信息学及组织表达分析[J]. 中国畜牧兽医, 2022, 49(3): 817-829.

SHI Yuzhu, LUO Rutang, CHEN Chao, WU Xu, LI Ang. Cloning, Bioinformatics and Tissue Expression Analysis of GDF9 Genes in Muscovy Duck[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 817-829.

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