中国荷斯坦牛HSF1基因microRNA SNPs 与耐热性能的相关性研究

中国农业科学 ›› 2011, Vol. 44 ›› Issue (3): 570-578 .

中国荷斯坦牛HSF1基因microRNA SNPs 与耐热性能的相关性研究

李秋玲,鞠志花,贾祥捷,黄金明,李建斌,李荣岭 ,李芳,王长法,仲跻峰

（山东省农业科学院奶牛研究中心）

收稿日期:2010-06-10 修回日期:2010-08-09 出版日期:2011-02-01 发布日期:2011-02-01
通讯作者: 王长法,仲跻峰

Identification of MicroRNA SNPs of HSF1 Gene and Their Association with Heat Tolerance in Chinese Holstein

LI Qiu-ling,JU Zhi-hua, JIA Xiang-jie, HUANG Jin-ming, LI Jian-bin, LI Rong-ling, LI Fang,WANG Chang-fa, ZHONG Ji-feng

（山东省农业科学院奶牛研究中心）

Received:2010-06-10 Revised:2010-08-09 Online:2011-02-01 Published:2011-02-01
Contact: WANG Chang-fa, ZHONG Ji-feng

摘要/Abstract

摘要：

【目的】分析638头中国荷斯坦牛HSF1基因多态性与耐热性的关系,以期为中国耐热奶牛分子育种提供参考依据。【方法】设计引物扩增HSF1基因,通过DNA测序、PCR-SSCP和PCR-RFLP技术筛查SNPs并分型,利用miRBASE数据库定位microRNA SNPs,利用SHEsis软件进行配对连锁不平衡分析和单倍型构建,利用SAS软件对不同基因型和单倍型组合个体耐热指标包括红细胞钾浓度、产奶量下降率、直肠温度和耐热系数进行相关分析。应用荧光定量RT-PCR技术研究热应激状态下HSF1 mRNA的转录水平。【结果】发现2个microRNA种子序列新 SNPs,T909C和G4693T。其中909位碱基距离microRNA种子序列5'端3 bp,4 693位突变直接使microRNA种子序列破坏。909位点CC、CT基因型奶牛耐热性能显著高于TT基因型（P＜0.05）;4693位点TT基因型奶牛耐热性能显著高于GG基因型（P＜0.05）。共构建出4种单倍型、发现10种单倍型组合。其中H2H4单倍型组合红细胞钾浓度和直肠温度显著低于H1H3单倍型组合（P＜0.05）,耐热系数显著高于H1H3组合（P＜0.05）,H2H4组合个体产奶量下降率显著低于H1H1组合个体（P＜0.05）,H2H4为耐热单倍型组合。热应激状态下HSF1 mRNA的转录水平在不同组织中存在差异,在心脏中最高,是肌肉中的11.24倍（P＜0.05）。【结论】HSF1基因microRNA SNPs可作为奶牛抗热应激的候选分子标记应用于育种实践。

关键词: 中国荷斯坦牛, HSF1, 耐热性能, microRNA, 多态性

Abstract:

【Objective】 Heat shock transcription factor1 (HSF1) is the key protein in regulating heat stress response. 【Method】The polymorphisms of HSF1 gene and their association with heat tolerance in 638 Chinese Holstein cattle were investigated for the purpose of providing molecular marker information to facilitate the breeding efficiency of thermo tolerant cows. Primers were designed to amplify HSF1 gene fragment. DNA sequencing, PCR-SSCP and PCR-RFLP methods were used to analyze the polymorphisms. MicroRNA SNPs of HSF1 gene were localized by blasting to miRBASE database. Linkage disequilibrium and haplotype were analyzed using SHEsis software. The association of these polymorphisms with heat tolerance index including potassium content in erythrocytes (PCE), decrease rate of milk production (R), rectal temperature (RT) and heat-tolerance coefficient (HTC) was analyzed by SAS software. The transcriptional level of HSF1 mRNA under heat stress conditions was studied by fluorescent quantitative RT-PCR technology. 【Result】 Two novel microRNA SNPs, T909C and G4693T, were found. The cows with CC and CT genotype showed higher heat tolerance than those with TT genotype at T909C locus (P＜0.05). The cows with TT genotype showed higher heat tolerance than those with GG genotype at G4693T locus (P<0.05). Four haplotype and 10 haplotype combinations were found. The cows with H2H4 haplotype combination had lower PCE, RT and higher HCT than those with H1H3 haplotype combination (P<0.05). The cows with H2H4 haplotype combination had lower R than those with H1H1 haplotype combination (P<0.05). H2H4 was the heat tolerant haplotype combination. The transcriptional level of HSF1 mRNA under heat stress conditions was different in different tissues. The highest is that in heart, which is 11.24-fold enrichment than that in muscle (P<0.05). 【Conclusion】 microRNA SNPs of HSF1 gene could be chosen as the molecular markers for choosing thermo tolerant dairy cattle in breeding program.

Key words: Chinese Holstein, HSF1, thermal tolerance, microRNA, polymorphism

李秋玲,鞠志花,贾祥捷,黄金明,李建斌,李荣岭 ,李芳,王长法,仲跻峰 . 中国荷斯坦牛HSF1基因microRNA SNPs 与耐热性能的相关性研究[J]. 中国农业科学, 2011, 44(3): 570-578 .

LI Qiu-ling,JU Zhi-hua,JIA Xiang-jie,HUANG Jin-ming,LI Jian-bin,LI Rong-ling,LI Fang,WANG Chang-fa,ZHONG Ji-feng
. Identification of MicroRNA SNPs of HSF1 Gene and Their Association with Heat Tolerance in Chinese Holstein
[J]. Scientia Agricultura Sinica, 2011, 44(3): 570-578 .

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