摘要
本论文对正在选育的地方优质鸡新品系二郎山山地鸡进行了不同饲养方式的饲养试验,对其脂肪性状进行了记录和测定,探索不同饲养方式对鸡肉质的影响；同时对其固醇调控元件结合转录因子家族成员SREBF1和SREBF2基因进行了全编码区序列测定和相关生物信息学分析；对不同饲养方式下,不同时间点以及不同组织的SREBF1和SREBF2基因表达模式进行了探索；对SREBF2基因的SNP位点进行了扫描和屠体性状的关联性分析,试验结果如下：
性能测定发现,8、10、13周龄的笼养、平养以及散养二郎山山地鸡的部分脂肪性状、血液生化指标和胸肌脂肪酸组成均存在显著或极显著(P<0.05或P<0.01)差异,提示这些性状或指标可能会因饲养方式的不同而受到影响。
克隆获得了二郎山山地鸡固醇调控元件结合转录因子家族成员SREBF1和SREBF2基因的全编码区序列,并翻译获得其氨基酸序列(1115aa和952aa)。通过生物信息学分析表明：SREBP-1和SREBP-2蛋白都不是跨膜蛋白,但存在跨膜区,同时都具有bHLH转录因子家族典型的保守结构功能域,说明了SREBP-1和SREBP-2蛋白均属于核转录因子家族。
采用SYBR green I定量PCR分析SREBF1和SREBF2基因表达时发现,同一种饲养方式下SREBF1和SREBF2基因在二郎山山地鸡的肝、腹脂、皮脂、胸肌、腿肌和尾脂腺中都有表达,而且不同基因在不同组织中表达的优势时间点不同。肝、尾脂腺和胸肌为SREBF1和SREBF2基因的优势表达组织。不同饲养方式下肝中的SREBF1基因表达量存在显著差异,散养鸡表现出较低的表达水平；SREBF2基因在腹脂和胸肌中的表达量存在显著差异,腹脂中散养鸡的表达水平较低,胸肌中散养鸡有较高水平的表达。
本研究采用测序和PCR-SSCP的技术设计引物,扫描了二郎山山地鸡和四川大恒优质肉鸡共300个个体的SREBF2基因外显子序列和邻近内含子序列。结果显示：10个群体中发现10个单核苷酸突变位点,分别位于r1：51382660、rl:51382391、r1:51381392、r1:51376638、rl:51376226、r1:51375686、rl:51374255、r1:51373368、rl:51372941和r1：51370350,10个突变位点的优势等位基因依次为A、C、A、A、C、C、G、G、G和C。由于SNP6、SNP8、SNP9三个位点的优势等位基因和基因型极其显著,且PIC<0.1,故将其剔除不构建单倍型。其余7个SNP可构成30种单倍型,获得117种复合单倍型,其中单倍型H4(A-C-G-A-T-A-G)、H10(A-C-A-A-C-G-C).H13(A-C-A-A-T-G-C)和H16(A-C-A-G-C-G-C)为优势单倍型。标记-关联分析发现,SREBF2基因SNP1对全净膛重的遗传效应达到极显著水平(TypeⅢSS,F=5.605,P=0.004)；SNP2对全净膛重的遗传效应达到显著水平(TypeⅢSS,F=3.418,P=0.034)；SNP4对活重的遗传效应达到显著水平(TypeⅢSS,F=3.881,P=0.022),对屠体重、半净膛重、全净膛重、胸肌重和腿肌重的遗传效应达到极显著水平(Type III SS,F=5.805,P=0.003；TypeⅢSS,F.6.303,P=0.0002；TypeⅢSS,F=6.841, P=0.001; TypeⅢSS,F=4.987,P=0.007;TypeⅢSS,F=6.576,P=0.002); SNP3.SNP5.SNP7和SNP10对屠体性状的遗传效应均未达到显著水平(P>0.05)。而单倍型分析发现,单倍型对胸肌重的遗传效应达到极显著水平(TypeⅢSS,F=1.966,P=0.006),对其它屠体性状的遗传效应均没有达到显著水平(P>0.05)。单倍型H21 H21是活重、屠体重、半净膛重、全净膛重和腿肌重的优势单倍型,H13H21是胸肌重的优势单倍型,而H4H21是皮脂厚的劣势单倍型,提示单倍型H21(A-T-G-A-C-G-C)对于提高肉鸡的生产性能有正向作用。
In this research, we carried out different feed-style tests to a new line which breeds Erlang mountainous chickens, by recording and testing the production and the traits of meat quality, we explored the effect of different feed-styles. Meanwhile, we cloned the entire coding sequence of two members for SREBFs, which were SREBF1 and SREBF2 and the correlations of the bioinformatics analysis were done. For different feed-styles, we also tested and analyzed the expression pattern of SREBF1 and SREBF2 in different growth points and in different tissues using SYBR Green I qRT-PCR. We also detected the SNPs of SREBF2 genes using PCR-SSCP and sequence screening methods in six stains of Daheng high-quality chicken in Sichuan province and Erlang mountainous chickens, and related analysis was conducted, the results are as follows:
Determination of properties found that at 8、10 and 13 weeks, the part of the fat traits、blood biochemical parameters and chest fatty acids, respectively, in caged feed chickens、indoor floor chickens and outdoor access chickens were significantly or extremely significantly (P<0.05 or P<0.01) different, suggested that these traits or indicators may be due to the different feed-styles.
We successfully obtained the full coding sequence of SREBF1 and SREBF2. Based on these two cDNA sequences, their amino acid sequences were increased (1115aa and 952aa). By bioinformatics, we found SREBP-1 and SREBP-2 were not transmembrane protein but contain membrane region, and conservative structural functional domain of bHLH transcription factor family. It implied that they all belonged to the bHLH family.
We used the real-time PCR (SYBR green I) method to test the expression level of SREBF1 and SREBF2 gene in different tissue including liver, abdomen fat, sebumcutaneum fat, breast muscle, leg muscle, and uropygial gland in at certain growth point of chicken with different feed-styles. The results indicated that SREBF1 and SREBF2 genes had expressed in all tissues at all growth points and there were growth points and tissues with higher expression level. We also found the expression in different feeding conditions was different. Liver, breast and uropygial gland are advantageous expressed tissues for SREBFl and SREBF2. In different feeding-styles, the expression level of SREBF1 in liver had significant differences (P<0.05), and free-range chicken had lower expression level. For SREBF2, there were significantly different expressions in abdomen fat and breast muscles (P<0.01), and the expression level in abdomen fat with outdoor access was lower, while in breast muscle the outdoor access had higher expression level.
In this study, we screened part sequences of extrons and intron of SREBF2 gene by sequencing and PCR-SSCP method. Ten SNPs were identified, including rl: 51382660, r1:51382391, r1:51381392, r1:51376638, r1:51376226, r1:51375686, r1:51374255, r1:51373368, r1:51372941 and r1:51370350, respectively. And the superiority alleles were A, C, A, A, C, C, G, G, G and C, respectively. Because SNP6, SNP8 and SNP9 had the extremely significant superiority alleles and genotypes, and the PIC＜O.1, so we deleted their haplotypes. Then 30 haplotypes were constructed to form by the other seven SNPs, and there were 117 composite haplotypes. H4 (A-C-G-A-T-A-G) H10 (A-C-A-A-C-G-C), H13 (A-C-A-A-T-G-C), and H16 (A-C-A-G-C-G-C) were advantageous haplotypes. By mark-associated analysis, we found SNP1 had extremely significant genetic effect on eviscerated weight (Type III SS, F=5.605, P=0.004), SNP2 had significant genetic effect on eviscerated weight (Type III SS, F=3.418, P=0.034). SNP4 had significant genetic effect on live-weight (Type III SS, F=3.881, P=0.022), and had extremely significant effect on carcass weight, semi-eviscerated weight, breast muscle weight and leg muscle weight (Type III SS, F=5.805, P=0.03; Type III SS, F=6.303, P=0.0002; Type III SS, F=6.841, P=0.001; Type III SS, F=4.987, P=0.007; Type III SS, F=6.576, P=0.002). SNP3, SNP5, SNP7 and SNP10 all had no significant genetic effect on carcass traits (P>0.05). Based on haplotype analysis, we found that haplotype had extremely significant genetic effect on breast muscle weight (Type III SS, F=1.966, P=0.006), but had no significant genetic effect on other carcass traits (P>0.05). H21H21 was superior haplotype of live-weight, carcass weight, semi-eviscerated weight, eviscerated weight, and leg muscle weight, H13H21 was for breast muscle weight, while H4H21 was inferior haplotype for skinfold thickness. So it can be concluded that H21 (A-T-G-A-C-G-C) may have positive effect on promoting the production performance for boiler chicken.
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