填饲对朗德鹅SCD5、SREBP2和ELOVL6甲基化和mRNA表达的影响

DNA甲基化是表观遗传学的研究热点之一.为了探索填饲是否能够引起朗德鹅Anser anser)肝脏脂类代谢中DNA甲基化水平以及基因表达量的变化和DNA甲基化修饰作用和基因表达之间的关系.本研究运用焦磷酸测序技术分析鹅肝脏硬脂酞辅酶A去饱和酶5基因(stearovl-CoA desaturase 5,SCD5)、固醇调节元件结合蛋白2基因(sterol regulatory element-binding protein 2,SREBP2)和超长链脂肪酸延伸酶6基因(elongase of very long chain fatty acids 6,ELOVL6)的甲基化水平,再运用qRT-PCR技术进行定量验证.结果显示,SCD5、SREBP2和ELOVL6各2个片段的甲基化水平均在20％以上,均属于高甲基化状态;对照组3个基因各2个片段的甲基化水平均高于填饲组,填饲组与对照组的SCD5-1S和SREBP2-1S片段甲基化差异显著(P＜0.05),SREBP2-2S片段甲基化差异极显著(P＜0.01),SCD5-2S、ELOVL6-1S和ELOLV6-2S片段甲基化差异不显著(P＞0.05);填饲组SCD5和ELOVL6的mRNA表达量均高于对照组,而SREBP2在填饲组的mRNA表达量显著低于对照组(P＜0.05).综合各项指标,填饲引起鹅肝脏组织SCD5、SREBP2和ELOVL6甲基化水平降低,SCD5和ELOVL6 mRNA表达量上升,SREBP2的mRNA表达量降低,表明SCD5和ELOVL6的甲基化修饰对mRNA的表达起反向调控的作用.本实验结果为表观遗传学上DNA甲基化研究方法提供参考,为选育鹅肥肝高产高质系提供重要的分子遗传学依据,也为人类肥胖症和脂肪肝等代谢疾病的研究提供理论依据.     

Effect of sire and sex on the intramuscular fatty acid profile and indices for enzyme activities in pigs

In this study, the effect of sire, sex and intramuscular fat content on the intramuscular fatty acid profile, in particular the long chain polyunsaturated fatty acids (PUFA) was investigated. Therefore, pork samples of the Longissimus thoracis were taken from 121 females and castrates that were the progeny of 5 boars. All animals had been fattened on the same diet and were slaughtered at a live weight of approximately 110 kg. Indices for the activities of Δ9, Δ6 and Δ5 desaturase, as well as elongase activity were estimated from ratios of product to precursor fatty acids. Intramuscular fat content was positively related to the total saturated fatty acid proportion (r = 0.376; p < 0.01) and the total monounsaturated fatty acid proportion (r = 0.579; p < 0.01), and inversely correlated with the total PUFA proportion (r = − 0.637; p < 0.01). A significantly higher index for Δ5 and Δ6 desaturase and elongase activity for PUFA metabolism was observed in females compared to castrate males. Sire had a significant effect on the intramuscular fatty acid profile, notably on the total n − 3 PUFA, and on most individual long chain n − 6 and n − 3 PUFA. The cis-9 C18:1/C18:0 index for Δ9 desaturase activity and the cis-11 C18:1/cis-9 C16:1 elongase activity index, as well as the combined desaturase and elongase enzyme activities in both the n − 6 and n − 3 PUFA chains were significantly influenced by sire.     

奶山羊ELOVL6基因启动子的克隆及活性区域分析

超长链脂肪酸延伸酶6(elongase of very long chain fatty acids 6,ELOVL6)主要催化C12～C16饱和或单不饱和脂肪酸的延伸,是长链脂肪酸延长反应的限速酶.本研究根据云南黑山羊(Capra hircus)的基因组序列(Gene ID:NW_005100691.1)设计引物,以血液DNA为模板,利用PCR技术扩增得到西农萨能奶山羊(C.hircus)ELOVL6基因启动子的全长序列,通过缺失分析,构建了10个包含ELOVL6启动子不同缺失片段的荧光素酶报告基因载体,与海肾荧光素酶对照报告基因载体(pRL-TK)共同转染西农萨能奶山羊乳腺上皮细胞,利用双荧光素酶系统检测不同片段的启动子活性.结果表明,克隆得到ELOVL6基因启动子全长序列2 370 bp(包含转录起始位点上游2 168 bp),其与牛(Bos taurus)、人(Homo sapiens)和鼠(Mus musculus)的基因组序列同源性分别为94％、81％和80％,LO VL6启动子上无典型的真核生物启动转录元件TATA框.缺失突变研究发现,ELOVL6基因启动子前端存在负调控元件,启动子核心区域位于转录起始位点上游109～40 bp,该序列包含过氧化酶体增殖物激活受体(peroxisom prolifeator-activated receptor,PPAR)、肝X受体(liver X receptor,LXR)、胆固醇调节元件结合蛋白-1 (sterol-regulatory element binding protein-1,SREBP-1)、特异性蛋白1(specificity protein 1,Sp1)及核因子Y(nuclear factor Y, NF-Y)等转录因子的结合位点,本研究为ELOVL6基因启动子的功能研究提供理论依据.     

Action mechanism of a novel herbicide, pyroxasulfone

The mechanism of action of pyroxasulfone was studied by examination of the inhibitory effects of this herbicide on the biosynthesis of very-long-chain fatty acids (VLCFAs) both in vivo and in vitro. Pyroxasulfone treatment drastically reduced the biosynthesis of VLCFAs and caused a buildup of fatty acid precursors in cultured rice cells. Pyroxasulfone specifically inhibited the elongation steps from C18:0 to C20:0, C20:0 to C22:0, C22:0 to C24:0, C24:0 to C26:0 and C26:0 to C28:0, catalyzed by VLCFA elongases (VLCFAEs) in plants including rice. These results suggested that pyroxasulfone is a potent inhibitor of VLCFA biosynthesis, and should be categorized within the K3 group of herbicides. Twenty putative VLCFAEs of rice were identified by blastp search with the amino acid sequences of Arabidopsis VLCFAEs. Oligo microarray and real time RT-PCR analysis revealed that Q5Z6S3 and Q8H7Z0, which were identified by their Uniplot ID number, might play important roles during the biosynthesis of C28:0 and C30:0 VLCFAs in shoot formation, and biosynthesis of C20:1 and C22:1 VLCFAs in cell proliferation, respectively. These VLCFAEs are likely targets for pyroxasulfone.     

超长链多不饱和脂肪酸在棉花中的异源合成

从球等鞭金藻、眼虫、高山被孢霉和拟南芥中分别克隆到Δ9链延长酶、Δ8去饱和酶、Δ5去饱和酶和Δ15去饱和酶基因,利用我们的多基因聚合方法,将这4个基因聚合到植物表达载体pCambia2300上,其中每个基因都含有独立的CaMV35S启动子和Tnos终止子。利用农杆菌介导法将该表达载体转入棉花,通过卡纳霉素和PCR筛选获得转基因阳性植株,提取转基因阳性植株叶片总脂肪酸,用气相色谱分析法检测到花生四烯酸(AA,20:4Δ5,8,11,14)和二十碳五烯酸(EPA,20:5Δ5,8,11,14,17)含量分别达1.0%和5.0%。表明通过基因代谢工程在棉花中异源合成EPA是可行的,为进一步在棉籽中生产VLCPUFAs奠定了基础。