胆囊收缩素A型受体基因序列变异和表达与略阳乌鸡饲料转化率的关联性研究

doi:10.3864/j.issn.0578-1752.2022.22.017

摘要/Abstract

摘要：

【背景】 食欲和饲料转化率的提高构成了现代肉鸡快速生长的生理基础。胆囊收缩素A型受体（CCKAR）是介导胆囊收缩素（CCK）饱食信号和消化调节作用的关键受体。鉴于CCK信号在能量平衡调节中的重要作用，CCKAR的表达水平与鸡的生长性能密切相关。然而，CCKAR水平调节鸡生长的确切机制尚不清楚。【目的】 通过研究阐明CCKAR基因表达水平和序列变异与鸡饲料转化率的关系，为鸡生长性状遗传调控研究和饲料转化率选育奠定基础。【方法】 测定略阳乌鸡12—16周龄料肉比（公鸡n=62，母鸡n=67）和26—30周龄料蛋比（n=150）。用PCR-RFLP法检测CCKAR基因内两个同义突变（g.73206714T>C和g.73209189C>T）的基因型。以性别为固定效应，用双因素方差分析检验g.73206714T>C和g.73209189C>T与料肉比的关联性，用单因素方差分析检验2个SNP与料蛋比的关联性。用JASPAR数据库预测g.73206714T>C对转录因子结合位点产生的影响。以g.73206714T>C为标记，选取10只杂合子（CT）公鸡，用PCR-RFLP法检测CCKAR基因在g.73206714T>C位点等位基因间的表达差异。选取30只带有采食量和料肉比记录的公鸡，用qPCR检测CCKAR基因在下丘脑、脑垂体、胰腺、胆囊和十二指肠中的表达水平，分析CCKAR表达水平与采食量和料肉比的相关性。【结果】 性别效应对略阳乌鸡料肉比有显著影响（F=29.44，P<0.0001），公鸡的料肉比（5.41±0.76）显著低于母鸡（6.62±1.00）。在两个同义突变中，只有g.73206714T>C与料肉比显著关联（F=8.44，P=0.0004），其中，TT型的料肉比（5.18±0.63）显著低于CC（6.37±1.1）和CT型（6.17±0.95）。CC型和CT型的料肉比无显著差异。g.73206714T>C与性别的互作效应对料肉比的影响不显著。G.73206714T>C对28个转录因子结合位点有潜在影响。等位基因特异性表达试验显示，T等位基因的转录本丰度显著高于C等位基因，表明CCKAR在等位基因间的表达差异很可能是构成g.73206714T>C与料肉比关联性的遗传基础。略阳乌鸡26—30周龄的平均料蛋比为2.85±0.41。g.73206714T>C和g.73209189C>T的基因型效应对料蛋比的影响均不显著。在5个组织中，CCKAR基因在胰腺中的表达水平最高，且与采食量呈负相关（r=-0.45, P=0.017）。但是，在其他组织中，CCKAR的表达水平与采食量的关联性均不显著。对于料肉比性状，CCKAR基因在胰腺（r=-0.41，P=0.03）和脑垂体（r=-0.57, P=0.0018）中的表达水平越高，料肉比越低。在其他组织中，这种负相关性均不显著。【结论】 研究发现CCKAR基因表达水平上调有利于提高鸡的饲料转化率。G.73206714T>C的T等位基因是一个与CCKAR基因高表达活性和高饲料转化率显著关联的分子标记。

关键词: 鸡, 生长, CCKAR, 饲料转化率, 食欲

Abstract:

【Background】 Increased appetite and feed conversion efficiency forms a physiological basis underlying high growth of modern commercial broiler chickens. Cholecystokinin A receptor (CCKAR) is a key receptor for mediating CCK signals and involving in regulation of appetite and digestion. In view of the important role of CCK signals in the control of energy balance, the levels of CCKAR expression are closely related to the growth of chickens. However, the mechanism by which the abundance of CCKAR affects the growth of chickens remains unclear. 【Objective】 The aim of this study was to elucidate the relationship of levels of CCKAR expression and sequence variants with feed conversion efficiency, which would promote the understanding of genetic basis of the growth traits and breeding for improvement of feed efficiency in the chicken. 【Method】The feed to meat conversion ratio of Lueyang black-boned chickens (rooster n=62, pullet n=67) from 12 to 16 weeks of age and the feed to egg conversion ratio from 26 to 30 weeks of age (n=150) were individually measured. Two synonymous mutations (g.73206714T>C and g.73209189C>T) in the CCKAR gene were genotyped by using PCR-RFLP. Association of two SNP with the feed to meat conversion ratio was tested by using two-way analysis of variance with sex treated as a fixed effect. Association of two SNP with the feed to egg conversion ratio was tested by using one-way analysis of variance. Disrupting effect of g.73206714T>C on transcription factor binding sites was predicted in silico by using JASPAR database. Ten birds that were heterozygotes (CT) at the g.73206714T>C locus were selected to analyze imbalance of CCKAR expression between T and C alleles. The difference of allele-specific expression was detected by using PCR-RFLP. The Levels of CCKAR expression were detected by qPCR in the hypothalamus, pituitary, pancreas, gallbladder and duodenum of Lueyang black-boned chickens (n=30). The correlation of levels of CCKAR expression with food intake and feed to meat conversion ratio was analyzed. 【Result】 Sex had a significant (F=29.44，P<0.0001) effect on the feed to meat conversion ratio, and the feed efficiency (5.41±0.76) of roosters was significantly superior to pullets (6.62± 1.00). Of two SNP, only g.73206714T>C was significantly associated with the feed to meat conversion ratio. The feed to meat conversion ratio (5.18±0.63) of the TT genotype was significantly lower than ones of CC (6.37±1.1) and CT (6.17±0.95) genotypes. The feed to meat conversion ratio had no significant difference between CC and CT. There was no significant interaction effect between genotypes of g.73206714T>C and sex. G.73206714T>C potentially disrupted binding sites of 28 transcription factors. Allele-specific expression assay showed that the abundance of transcripts with the T allele was higher than one of transcripts with the C allele, supporting that differential expression of CCKAR gene between alleles might likely contribute to genetic association of g.73206714 with the feed to meat conversion ratio. Average feed to egg conversion ratio of Lueyang black-boned chickens was 2.85±0.41 at the 26-30 weeks of age. Both g.73206714T>C and g.73209189C>T had no significant effect on the feed to egg conversion ratio. The levels of CCKAR expression in the pancreas were the highest among five tissues, which showed a negative correlation (r=-0.45, P=0.017) with feed intake. But there was no significant correlation between levels of CCKAR expression and feed intake in other tissues. In the pancreas (r=-0.41, P=0.03) and pituitary (r=-0.57, P=0.0018), the higher levels of CCKAR expression was, the lower the feed to meat conversion ratio was. But in other tissues, there was no the negative correlation.【Conclusion】 This study found that increased levels of CCKAR was associated with high feed efficiency. The T allele of g.73296714T>C was associated with high expression activity of CCKAR gene, and with high feed efficiency.

Key words: chicken, growth, CCKAR, feed conversion efficiency, appetite

王哲鹏,周雯馨,贺俊锡,虎巧燕,赵家悦. 胆囊收缩素A型受体基因序列变异和表达与略阳乌鸡饲料转化率的关联性研究[J]. 中国农业科学, 2022, 55(22): 4539-4549.

WANG ZhePeng,ZHOU WenXin,HE JunXi,HU QiaoYan,ZHAO JiaYue. Association of Levels of Cholecystokinin A Receptor Expression and Sequence Variants with Feed Conversion Efficiency of Lueyang Black-Boned Chicken[J]. Scientia Agricultura Sinica, 2022, 55(22): 4539-4549.

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