苦参碱对奶牛乳腺上皮细胞增殖、凋亡及抗氧化能力的影响

【目的】探究苦参碱对体外培养的奶牛乳腺上皮细胞(BMECs)增殖、凋亡及抗氧化能力的影响。【方法】利用含0(A组),25(B组),50(C组),75(D组)和100μg/mL(E组)苦参碱的培养基培养奶牛乳腺上皮细胞。通过四甲基偶氮唑盐(MTT)法检测BMECs活性,采用流式细胞仪(AnnexinV/PI双染法)检测苦参碱对BMECs凋亡的影响,并检测苦参碱对BMECs抗氧化酶活性及丙二醛(MDA)含量的影响,采用real-time PCR对BMECs中Caspase-3、p53、STAT1和SOCS3基因的相对表达量进行检测。【结果】用药5d时,低质量浓度(25和50μg/mL)苦参碱对BMECs增殖具有促进作用,高质量浓度(75和100μg/mL)苦参碱对细胞增殖具有抑制作用;B~E组BMECs的凋亡率均极显著高于A组(P0.01);B~E组BMECs培养上清液中NO和乳酸脱氢酶(LDH)水平明显高于A组。B~E组BMECs的过氧化氢酶(CAT)活性均比A组高,其中C组极显著高于A组(P0.01);B~E组的谷胱甘肽过氧化物酶(GSH-Px)活性均极显著高于A组(P0.01),E组的超氧化物歧化酶(SOD)水平极显著高于A组(P0.01),各组MDA含量无显著性差异。与A组相比,苦参碱上调了B~E组BMECs中Caspase-3、p53、STAT1和SOCS3基因的相对表达量。【结论】低质量浓度苦参碱能够促进BMECs增殖,高质量浓度苦参碱则会抑制BMECs增殖;不同质量浓度苦参碱均可提高BMECs的抗氧化能力,其中50μg/mL苦参碱提高BMECs抗氧化能力的效果最明显。     

羊IL-1Ra基因全长cDNA克隆及生物信息学分析

试验旨在对羊白介素1受体颉颃因子（interleukin-1 receptor antagonist，IL-1Ra）基因进行全长cDNA克隆及生物学分析。根据布鲁氏菌感染羊白细胞层SSH cDNA文库中的IL-1Ra基因序列信息及已知核苷酸序列（GenBank登录号：KC425613.1）设计引物，利用RT-PCR结合RACE方法扩增并克隆IL-1Ra基因，对其进行序列及相关分子特性分析。结果表明，羊IL-1Ra基因全长为1 228 bp，可编码174个氨基酸，含有1个完整的IL-1保守结构域和1个IL-1Ra结构域（PHA02651结构域）。IL-1Ra分子质量为19 765.8 u，等电点（pI）为5.72，其分子式为C₈₈₅H₁₃₈₅N₂₃₅O₂₅₆S₁₁，且含有信号肽。二级结构分析显示，IL-1Ra分子存在较多的β折叠及受体结合位点，与三级结构预测结果一致，且与人/鼠IL-1Ra分子的空间结构相似度极高，达到90%以上。羊IL-1Ra有IL-1特有的三叶草结构，其酶结合结构域位于TYR⁴⁷至GLU⁶⁶之间。将羊IL-1Ra与牛、虎鲸、宽吻海豚、猪、家犬、人、鼠等14个物种进行蛋白质同源性比对，发现在各物种间存在5个高度同源的半胱氨酸位点。系统进化树分析发现，羊IL-1Ra基因全长cDNA所编码的氨基酸序列同山羊、牛单独形成一个分支。本研究结果为今后深入研究IL-1Ra基因的生物学功能奠定了基础。     

122份栽培桃品种（系）黄白肉性状的分子标记辅助鉴定

【目的】 类胡萝卜素裂解双脱氧酶基因（Carotenoid Cleavage Dioxygenases 4,CCD4）控制桃果肉颜色（白/黄）,CCD4存在3种等位基因。本研究利用Indel、SSR荧光标记毛细管电泳及SNP鉴定等基因分型技术分析我国主要桃黄白肉品种（系）中CCD4等位基因的差异,为主要黄/白肉品种（系）的基因型鉴定、亲本选配和选择相应的分子标记对不同来源子代的果肉颜色进行鉴定奠定基础。【方法】利用已经报道的桃不同果肉颜色中CCD4等位基因3种突变类型,合成不同引物进行PCR扩增,LTR反转录转座子插入突变经1%的琼脂糖凝胶电泳检测,CT单元重复的PCR产物在ABI3730XL测序仪上进行SSR荧光标记毛细管电泳检测,SNP标记经Sanger测序后利用ContigExpress软件分析CCD4等位基因的碱基替换（A→T）。综合以上结果,统计每份材料中CCD4等位基因的突变类型与果肉颜色的一致性。【结果】通过对不同来源的122份桃品种（系）材料进行基因型分析,发现CCD4发生LTR反转录转座子插入突变材料的基因型共有31份,占总材料的25.4%,其中纯合插入突变材料的片段扩增长度为729 bp,共有8份,占总突变的25.8%;CCD4发生微卫星重复序列突变材料存在2 bp的插入,扩增片段长度为179 bp,该类型共有68份,占总材料的55.7%,其中纯合插入材料25份,占总突变的36.8%;CCD4发生A→T碱基替换突变的材料较少,仅有1份,占总材料的0.82%,实际应用中可以不考虑该种类型。CT和LTR插入的两种突变类型的黄肉品种（系）有7份,占总材料的5.7%。研究结果表明,LTR反转录转座子插入突变和微卫星序列重复突变是黄肉桃中CCD4等位基因的主要突变类型。其中CCD4发生一种纯合突变或两种杂合突变桃品种（系）为黄肉类型,分子标记鉴定结果与调查的122份桃品种（系）黄白肉表型性状完全一致,准确率为100%。【结论】采用分子标记明确了122个桃品种（系）黄/白肉性状的基因型,为不同亲本组合子代表型鉴定的标记类型选择提供了技术支撑,为建立桃种质材料黄/白性状的分子辅助育种体系和黄肉桃的选育奠定了基础。     

Characterization of Genomic Integration and Transgene Organization in Six Transgenic Rapeseed Events

To characterize the DNA rearrangement of both the T-DNA region and the genomic insertion site during T-DNA insertion, the Genomewalker strategy was used to isolate the junctions between the inserted DNA and the plant genomic DNA in six rapeseed events as well as the genomic DNA at the sites before integration. During transformation in each of the six events, portions of both the right border（RB） and left border（LB） regions of the T-DNA were deleted, ranging from a 7 nucleotide deletion of the LB repeats in event RF1 to a 207 bp deletion of the LB region in event RF2. For the six events, T-DNA integration resulted in a deletion at the target site spanning less than 100 bp. Sequence analysis indicated that the T-DNA was integrated into the coding region of various native rapeseed genes in events RF1 and RF2. Duplications of the genomic DNA target site were observed in events RF2, RF3 and Topas 19/2. And multimerization of transgenes was found in event Topas 19/2, in which, the T-DNA was integrated as a head-to-head（RB-to-RB） concatemer into the recipient genome. In event MS1, chromosomal translocation or a large target-site deletion may have occurred during T-DNA integration, which was identified due to a failure to amplify the presumptive insertion site based on the flanking rapeseed DNA sequences. Our results provide comprehensive data concerning transgene organization and the genomic context of the T-DNA in six rapeseed events, which can aid in the developing of insert fingerprinting and the monitoring of long-term genetic stability and potential unintended effects of transgenic events.     

草原生态奖补政策实施效应——基于政治社会学视角的实证分析

草原生态奖补政策有助于保护草原生态,促进畜牧业的发展转型,有利于民族地区的和谐稳定。生态奖补政策的实施对牧民生计、基层社会治理等方面也产生了一些负面影响。未来的政策实践过程中,需要建立政策评估反馈机制,不断提高生态奖补标准,完善奖补资金发放办法,着力调动地方政府积极性,力求实现奖补手段的多样化,不断提高政策的法制化、系统化水平。     

Effect Comparison of Adenovirus Vector Transfer into Different Buffalo Cells

Buffalo mammary epithelial cell,cumulus cell and fibroblasts were transfected by adenovirus vectors and compared their transfection efficiency.293 cells were transfected with pBHGloxdelE13cre and pDC316-eGFP by liposome,the virus was collected and titer was detected.Buffalo mammary epithelial cell,cumulus cell and fibroblasts were exposed to different multiplicity of infection (MOI) of adenovirus vectors.After 72 h,the cells were observed with inverted fluorescence microscope,and transfection efficiency was calculated.When the MOI was 25,50,100,200 and 400,the transfection efficiency of fibroblasts were 0.7%,7.0%,9.0%,12.5% and 34.0%,the transfection efficiency of cumulus cells were 42.5%,55.3%,57.4%,76.0% and 80.0%,the transfection efficiency of mammary epithelial cells were 88.7%,100%,100%,100% and 100%.The results showed that the transfection efficiency of mammary epithelial cell was the best,followed by cumulus cell,and fibroblast was poor.     

Effects of Different Nitrogen Treatments on N use Efficiency and Grain Yield of Mechanically-transplanted Hybrid Rice

In order to investigate effects of topdressing on grain yield and N use efficiency of mechanically-transplanted hybrid rice,this experiment was conducted with 2-line hybrid rice cultivars,Quanliangyou 1 and Quanliangyou 681 with 3 application levels of N fertilizer,No,N1,N2,respectively.The results showed that there were no significant differences in grain yields of the 2 varieties between treatments N1 and N2.It was indicated that N is not the factor which could limit grain yield within limited application levels of N fertilizer.High N fertilizer treatment could contribute to the formation of effective panicles and spikelets per panicle,but it could also lead to the decline in grain filling rate.It was suggested that lowering the amount of Nfertilizer could facilitate improvement of N-use efficiency.     

Cloning and Sequence Analysis of Buffalo Keap1 Gene and Investigation of Its Expression Pattern in Different Tissues

In this study,the CDS sequence of buffalo Keap1 gene was cloned and analyzed,then its expression pattern in different tissues was also investigated.A pair of primers of buffalo Keap1 gene was designed based on the nucleotide sequence of Bos taurus Keap1 gene from GenBank,and then the buffalo Keap1 gene was amplified.Using the bioinformation techniques,the gene sequence and the protein structure were analyzed.The expression level of Keap1 gene in different tissues were detected with Real-time quantitative PCR.The results showed that the length of buffalo Keap1 gene coding sequence was 1 875 bp and encoded 624 amino acids.The multiple sequence alignment results showed that buffalo Keap1 gene shared 99%,96%,92% and 90% of similar nucleotide sequence with that of Bos taurus,Ovis aries,Sus scrofa and Homo sapiens,respectively.And the phyogenetic tree also showed the conservatism between several different species.The second structure of buffalo Keap1 protein was predicted as 24 alpha regions,40 beta regions,38 turn regions and 27 coil regions.In addition,the results of Real-time quantitative PCR showed that Keap1 mRNA exists in all seven tissues,but the most abundant expression was in heart and the minimal expression was in liver and spleen.The results provided an foundation for further study of Keap1-Nrf2-ARE signal pathway,for enhancing the ability of antioxidant of buffalo embryo in vitro culture.