Effects of forage type,body condition and single‐nucleotide polymorphisms in the bovine cytochrome P450 regulatory region on cow productivity*

Single‐nucleotide polymorphisms (SNP) in the coding sequence of cytochrome p450 (CYP3A28) have been associated with milk yield and composition, and calving traits in cows. In this study, we aimed to determine whether (i) the CYP3A28 regulatory region was polymorphic and (ii) SNP genotype, forage type, body condition and their interactions affect cow productivity. Primers for CYP3A28 promoter were designed to amplify a 483‐bp segment by PCR. Amplicon sequences revealed seven SNP (T‐318C, T‐113A, C‐189T, T‐78G, A6G, G17A and T21C) in Brahman (38 cows), Brahman x Angus reciprocal crosses (47 cows) and crossbreds (98 cows). Angus cows (n = 41) appeared to be fixed at those SNP locations. Genotype and forage {endophyte‐infected tall fescue [KY+; Lolium arundinaceum (Schreb.) S. J. Darbyshire] vs. bermudagrass [Cynodon dactylon (L.) Pers.]} effects on lifetime (8‐years) calving rate, and calf weaning weights and heights were determined in Herd 1 (126 cows); genotype and BC (low vs. moderate) effects on calving date and calving percent were determined in Herd 2 (98 cows). Four SNP (T‐318C, T‐113A, A06G and T21C) appeared to be related to cattle productivity, CC cows at T‐318C having a lower (p < 0.05) lifetime calving rate than TC or TT cows (65%, 85% and 81% respectively). Cows that grazed KY+ and were TT at T‐318C produced calves that tended (p < 0.07) to weigh less than their contemporaries. Moreover, calves of TT cows were shorter (p < 0.05) at weaning than calves of CC or TC cows. In Herd 2, moderate‐BC cows that were TT or AA at T‐318C, T‐113A, T‐78G, A6G and T21C had greater (p < 0.05) calving rates (74–80%) than heterozygous cows (46–60%), and low‐BC cows that were AA at G17A calved at least 6 days earlier (p < 0.05) than heterozygous cows. Our findings suggest that SNP in the CYP3A28 regulatory region of Brahman‐influenced cows are associated with cattle productivity.     

日粮添加免疫生长促进剂C96对雏鸡若干血液指标的影响

选择１２０只１日龄ＡＡ商品健康雏鸡,随机分为４组,每组３０只。初步观察了日粮添加免疫生长促进剂Ｃ９６对血液中红细胞总数（ＲＢＣ）、比容（ＰＣＶ）、血红蛋白含量（Ｈｂ）、网织红细胞比例（ＲＣＣ）、白细胞总数（ＷＢＣ）等的影响。结果表明：肉雏鸡在１５ｄ时,日粮添加Ｃ９６为１１ｍｇ／ｋｇ,饲料的Ｃ９６组和疫苗－Ｃ９６组,与对照组和疫苗组比较,ＲＢＣ和Ｈｂ增高,ＷＢＣ显著下降,ＲＣＣ降低,ＰＣＶ无明显规律性变化;３０ｄ和４５ｄ时,添加Ｃ９６组和疫苗－Ｃ９６组的ＲＢＣ、Ｈｂ和ＰＣＶ,与对照组和疫苗组基本相近,ＷＢＣ和ＲＣＣ均比对照组和疫苗组低或明显低。但３０ｄ时疫苗组的ＷＢＣ比对照组高。整个试验期,ＲＢＣ、ＷＢＣ和ＰＣＶ随日龄的增长呈上升趋势,ＲＣＣ呈波动性下降。     

Promoter identification and effect on activation of NF-κB of porcine ISG58

Interferon (IFN)-stimulated gene (ISG) 56 family (composed of ISG54, ISG56, ISG58, and ISG60) plays important roles in defense against viral infection in mammalian cells. Numerous studies have been conducted on ISG54, ISG56, and ISG60; however, little is known on ISG58. In the present study, the upstream sequence of porcine ISG58 gene was first characterized as functional promoter by luciferase reporter assay, and then two directly adjacent IFN-stimulated response elements (ISREs), one at ?206 to ?194 (ISRE-I) and a second one, directly upstream of this element at ?219 to ?207?bp (ISRE-II), were identified using the bioinformatics method. The subsequent site-directed deletion and transient transfection experiments showed the candidate ISREs are functional. ISRE-I works better than ISRE-II and synergistic cooperation exists between two ISREs. Additionally, the effect of porcine ISG58 on activation of NF-κB was analyzed using the dual-luciferase reporter assay. The results will contribute to revealing the role of ISG58 in immune response.     

新牧1号苜蓿两个抗逆相关基因启动子的克隆及分析

采用实时荧光定量PCR分析了新牧1号苜蓿（Medicago varia Xinmu 1）MvNHX1和MvDREB1基因在盐胁迫下的表达情况。此外,根据已获得的MvDREB1和MvNHX1基因序列设计特异引物,并以新牧1号苜蓿的基因组DNA为模板,克隆得到了这两个基因的启动子。利用生物信息学方法,分析这两个基因启动子的类型和结构,结果表明,MvNHX1和MvDREB1基因的启动子序列中均含有通用启动元件和上游调控元件,如CAAT框、TATA框、光响应元件、低温响应元件等,但部分响应元件的种类和数量不同。通过对两个基因启动子的克隆、分析及比较为进一步研究这两个基因的表达调控机制奠定了基础。     

sikFBA4基因启动子的克隆及低温表达模式分析

天山雪莲生长于常年积雪覆盖高海拔处的高山区域,具有很强的极端低温生境适应能力,属于研究植物低温适应的良好模式植物。前期的研究表明,sikFBA4基因可以显著提高番茄的抗寒能力。为进一步研究sikFBA4基因的耐寒响应模式,以天山雪莲为材料,采用实时荧光定量PCR分析sikFBA4在低温胁迫下的表达模式;利用高效热不对称PCR法(Hi-Tail PCR)克隆sikFBA4启动子序列并进行生物信息学分析。为分析PsikFBA4序列克隆的完整性及转录表达特性,将PsikFBA4与GUS基因融合在烟草中进行瞬时表达。结果表明,sikFBA4在低温胁迫条件下的表达发生瞬时显著上调,并在1 h达到峰值,而后表达下调。通过启动子克隆分析,在PsikFBA4序列-1648 bp位置处具有冷响应元件LTRE;进一步的GUS染色和酶活力测定结果表明,低温能够显著提高GUS基因的表达活性,说明sikFBA4属于低温诱导型基因。SikFBA4基因启动子的克隆、序列分析以及表达分析,为进一步探究雪莲果糖-1,6-二磷酸醛缩酶(sikFBAs)基因的表达与调控机制奠定了基础。     

发酵促进型青贮添加剂的研究进展及其应用现状

青贮添加剂是提高青贮饲料品质的重要措施之一。近几十年来,人们对于添加剂在牧草青贮技术中的应用进行了大量的研究,而且取得了重要进展,并成为目前青贮技术发展的重点,其中发酵促进型添加剂是近年来研究的热点。本文对乳酸茵、纤维素酶、绿汁发酵液和糖类等发酵促进型牧草青贮添加剂的研究进行了综述,阐述了研究的现状,指出了存在的问题,并对未来的趋势进行了展望。     

基于四环素调控系统构建白蛋白启动子调控大鼠uPA转基因表达的慢病毒载体

基于四环素调控系统构建白蛋白(albumin,Alb)启动子调控大鼠uPA(urokinase-type plasminogen activator,ruPA)转基因肝特异性过表达的慢病毒载体pLVX-Alb-TetOne-TRE-ruPA-T2A-CopGFP(pLATTRUTG)。以CTG0875-2-11质粒为模板,PCR扩增大鼠的uPA(ruPA)基因,3’端添加Flag标签,In-Fusion克隆至pLVX-Alb-TetOne-TRE-T2A-CopGFP(pLATTTG)质粒中,得到慢病毒载体pLVX-Alb-TetOne-TRE-ruPA-T2A-CopGFP(pLATTRUTG),所构建质粒经测序和酶切鉴定。将pLATTRUTG瞬时转染293T细胞,转染后24 h倒置荧光显微镜检测CopGFP表达;接着向6孔细胞培养板内加入强力霉素(Doxycycline,Dox),48 h后在倒置荧光显微镜下观察CopGFP表达(包括未加Dox的孔),随后收集细胞以提取总RNA和总蛋白,用于RT-qPCR检测ruPA及报告基因表达和Western blot检测标签蛋白Flag表达。酶切和测序确证我们成功构建了慢病毒载体pLATTRUTG;瞬转293T细胞后,24 h倒置荧光显微镜下可见零星细胞(约占0.1%)发弱的绿色荧光,加Dox 48 h后所有细胞展现强的绿色荧光,而不加Dox的孔内仍然只见到零星细胞(约占0.1%)发弱的绿色荧光。RT-qPCR和Western blot检测结果显示,与不加Dox的细胞相比,加Dox的细胞中ruPA、报告基因CopGFP和Flag表达水平显著升高。结果提示,成功基于四环素调控系统构建Alb启动子调控大鼠uPA转基因表达的慢病毒载体pLATTRUTG,为相关后续实验奠定了基础。     

Sequence of the domestic duck (Anas platyrhynchos) growth hormone-encoding gene and genetic variation in the promoter region

The duck growth hormone encoding gene and its promoter region were amplified by polymerase chain reaction (PCR). A total of 5.25 kb were cloned and sequenced. Duck growth hormone (GH) consists of five exons and four introns and is structurally similar to mammalian and chicken GH gene. Although the distal region of duck GH promoter showed no similarity to chicken and turkey promoters, the proximal region of the promoter contained two putative Pit‐1 binding sequences, and showed similarity to chicken and turkey GH promoters. Genetic variation was detected at five positions of the promoter region. The results of this study indicate that the expression of duck GH is likely regulated in a similar manner to that of chicken GH via enhancer‐type cis‐acting elements and the presence of genetic variation in the duck GH gene may be applicable to marker‐assisted selection.     

鸡HSP90AA1基因SNP检测及启动子区CpG岛的甲基化研究

试验旨在获得鸡热休克蛋白90α(HSP90AA1)基因序列并分析其基因结构和相关遗传变异,检测HSP90AA1基因启动子区CpG岛的甲基化状态,初步探索HSP90AA1基因在肌肉组织生长发育中的作用。以文昌鸡和北京油鸡为试验材料,利用PCR扩增鸡HSP90AA1基因组序列;通过基因测序寻找该基因中的单核苷酸多态性(SNP)位点;使用在线软件MethPrimer预测鸡HSP90AA1基因中CpG岛的位置;应用MassArray质谱法检测鸡胸肌中HSP90AA1基因启动子区CpG岛的甲基化水平,比较分析文昌鸡和北京油鸡HSP90AA1基因的甲基化差异。结果显示,在鸡HSP90AA1基因组中共发现7个SNPs位点,分别位于启动子区(A-189G,C-109T)、第1外显子(A+6G)、第2外显子(C+343T)、第2内含子(A+634G、A+836G)和第7内含子(A+3449G);鸡HSP90AA1基因包含10个外显子和9个内含子,其启动子区存在1个CpG岛,位于-1 802～-469bp处;在HSP90AA1基因启动子区共检测了42个CpG位点的甲基化水平,文昌鸡和北京油鸡中分别有9个(CpG_16.17.18、CpG_21.22.23、CpG_32.33和CpG_57)和4个CpG位点(CpG_1、CpG_5.6和CpG_57)在胸肌生长发育过程中发生甲基化改变。结果表明,文昌鸡与北京油鸡HSP90AA1基因序列信息和启动子区CpG岛的甲基化水平不同,这可能导致两种鸡对于应激反应具有不同的耐受程度。以上试验结果将为文昌鸡和北京油鸡生长发育规律、系统选育等方面的研究提供表观遗传学依据。