利用iTRAQ蛋白质组技术筛选与鸡喙畸形相关的候选蛋白

目的]中国地方鸡种如北京油鸡、清远麻鸡存在喙畸形现象,表现为上下喙咬合不全,呈交叉状。严重影响鸡饮水和采食,从而影响个体发育和生产性能的发挥,造成一定经济损失。笔者根据喙畸形个体的系谱记录,发现喙畸形的形成受到遗传因素的影响,但具体机制尚不明确。蛋白是行使各种生物学功能的最终形式之一,喙畸形个体的发生可能是由于核心蛋白或者相关调控蛋白代谢异常造成的。利用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,i TRAQ)技术以及生物信息学分析方法,筛选鸡畸形喙与正常喙中差异表达的蛋白,作为喙畸形相关的重要候选蛋白,为进一步研究北京油鸡喙畸形的遗传机制奠定基础。方法]挑选3只120日龄喙畸形的公鸡作为试验组,编号为W1、W2、W3,同时,挑选与试验组个体为同胞关系(全同胞或半同胞)的喙正常公鸡作为对照组,编号为Z1、Z2、Z3。将喙畸形与其同胞正常个体作为一个比对组,i TRAQ试验具体包含3个比对组,即W1 vs Z1,W2 vs Z2,W3 vs Z3。屠宰个体后,剔除喙组织周围肌肉和筋膜,分离得到喙上颌骨和下颌骨,提取总蛋白样品,应用6个i TRAQ标签标记各蛋白样品,经过色谱层析预分离,联合液相串联质谱分析,采用Mascot 2.3.02软件对蛋白进行鉴定和定量分析。试验组(W)与其同胞对照组(Z)样本比较(W1 vs Z1,W2 vs Z2,W3 vs Z3),选择肽段数≥2,表达差异值1.2(上调)或0.83(下调),且P0.05的蛋白作为差异表达蛋白。结果]利用i TRAQ技术一共在喙组织中鉴定到3 372个蛋白,鉴定到的特异性肽段有12 769个。其中原鸡蛋白1 869个,分子质量主要分布在10—100 k D之间。3个比对组共鉴定出159个表达量有显著差异的蛋白质,其中包含70个表达上调的蛋白,89个表达下调的蛋白。统计各比对组蛋白表达差异值发现,表达量差异较大的上调蛋白质有LPL、MLC-2、CO9A1、MATN3、HSP90B1等,表达量差异较大的下调蛋白质有MBP、RLA1、PRVM、HAPLN1等。结合已经报道的这些蛋白的生物学功能,其中CO9A1、MATN3、HAPLN1与软骨合成和软骨骨化相关,CO9A1是带状软骨纤维的组成物质,MATN家族是非胶原性细胞外基质蛋白家族,HAPLN1是软骨细胞外基质的重要组成成分;PRVM是细胞内钙离子结合蛋白,参与调控Ca2+离子信号通路;LPL是多功能酶,主要在脂质代谢和转运过程中起作用,参与调控PPAR信号通路。初步筛选出CO9A1、MATN3、HAPLN1、PRVM、LPL作为与鸡喙畸形相关的候选蛋白。结论]差异表达蛋白的发现为鸡喙畸形形态的发生提供蛋白质水平上的理论依据。

iTRAQ-Based Proteomic Analysis for Identification of Candidate Proteins Underlying Beak Deformity in Chickens

【Objective】Beak deformity has been reported in some indigenous chickens like Beijing-You chickens and Qingyuan partridge chickens with deformed beaks have problems with eating and drinking resulting in obvious reductions of growth and production performances. Observations on pedigreed individuals with deformed beaks shed light on the heritability of this trait. The aim of this study was to identify the candidate proteins related to beak deformity using isobaric tags for relative and absolute quantitation (iTRAQ) technology and bioinformatic analysis. The results can build the basis for further understanding the mechanism of beak deformity in chickens. 【Method】Three chickens with deformed beaks (W1, W2 and W3) and their sibs (Z1, Z2 and Z3) of 120 days of age were used in this study. Beak sample proteins of these chickens were collected by using tissue total protein lysis buffer. Six iTRAQs were used to label samples of three contrast groups (W1 vs Z1, W2 vs Z2, and W3 vs Z3). After separation by chromatography and analysis of liquid chromatography tandem mass spectrometry, differentially expressed proteins were identified and quantitatively analyzed by Mascot 2.3.02 software. Comparing deformed samples with the normal samples, we chose some differentially expressed proteins which consisted of 2 or more peptides, P＜0.05, and the ratio of which was more than 1.2 (up-regulated) or less than 0.83 (down-regulated). 【Result】 In total, 3 372 proteins were detected from three contrast groups using iTRAQ and 1 869 of them were Jungle fowl proteins. Of these, 159 were differentially expressed proteins, 70 were up-regulated and 89 were down-regulated. The most up-regulated proteins included LPL, MLC-2, CO9A1, MATN3, and HSP90B1, while the most down-regulated proteins were MBP, RLA1, PRVM, and HAPLN1. Protein CO9A1, MATN3, and HAPLN1 were associated with cartilage synthesis and ossification. PRVM is an intracellular calcium binding protein involved in regulating the Ca²⁺ ion signaling pathway. LPL is a multifunctional enzyme and mainly plays a role in lipid metabolism and transport, which involves regulating PPAR signaling pathways. 【Conclusion】 Proteins CO9A1, MATN3, HAPLN1, PRVM, and LPL were proposed as candidate proteins for beak deformity. The discovery and identification of these proteins can offer a valuable insights into basis of protein level of beak deformity in chickens.