陆地棉REM基因家族全基因组鉴定及表达分析

[目的]REM(Reproductive meristem)基因家族编码一类转录因子在植物生长发育过程中发挥重要作用,但在棉花中未见报道.[方法]基于陆地棉基因组数据和公共数据库中的转录组数据,运用生物信息学方法对陆地棉REM基因家族成员进行系统鉴定,并对该基因家族的理化性质、基因结构、组织表达特异性以及胁迫条件下的表...     

半滑舌鳎vasa调控区的克隆、表达载体构建及其驱动活性检测

为研究半滑舌鳎(Cynoglossus semilaevis)vasa(Csvasa)基因调控区的功能,在已克隆的Csvasa基因编码序列的基础上,采用基因组步移和PCR扩增的方法克隆得到Csvasa调控区,通过生物信息学方法分析vasa基因5′区,并构建了含Csvasa基因调控区的绿色荧光蛋白(GFP)表达载体(p Csvasa-GFP-T),进一步通过显微注射技术初步验证调控区的驱动活性。结果表明,通过基因组步移和PCR扩增获得Csvasa 5′区5 166 bp和3′区1 655 bp,利用在线生物信息学软件对5′区序列进行分析,发现在转录起始点上游26 bp处存在保守的TATA框,以及潜在的转录因子结合点如SRY、Oct-1、Sox-5、CREB、GATA、AP-1、C/EBP、Sp-1、c-Myc、HNF、NKX2-5、V-Myb等。通过显微注射技术,将所构建的p Csvasa-GFP-T表达载体注射于青鳉(Oryzias latipes)受精卵并进行培养观测,发现Csvasa调控区能够驱动GFP在青鳉胚胎内表达,荧光表达率为81%。将有荧光的胚胎培养为成鱼,检测外源基因的整合率为11.5%。这些结果为进一步研究半滑舌鳎原始生殖细胞(PGCs)的标记、追踪和操作研究以及半滑舌鳎的性别控制等奠定了基础。     

马铃薯地上部绿色组织中糖苷生物碱合成调控的研究

为提高马铃薯品种(系)地上部分糖苷生物碱(SGAs)的含量,增强其抗逆性并改良块茎的品质。克隆了马铃薯茄啶鼠李糖基转移酶基因(sgt3)cDNA和1,5–二磷酸核酮糖羧化酶小亚基基因启动子(rbcS P);对sgt3亚细胞定位预测显示,该蛋白不具有叶绿体转运肽、线粒体导肽和分泌信号肽序列,推测可能位于细胞质;将克隆的sgt3 cDNA片段及rbcS重组到pCEPSP载体上,构建了具有草甘膦抗性标记的绿色组织特异表达sgt3基因植物表达载体。通过农杆菌介导法对马铃薯品种‘陇薯3号’和‘夏波蒂’进行转化,共获得了12株抗草甘膦的阳性转基因植株。对转基因植株的目的基因表达水平和SGAs含量分析发现,地上部sgt3基因相对表达量较未转化植株提高1.3~3.0倍,SGAs含量增加20%~37%,而转基因植株的块茎中SGAs含量变化不显著。本研究的结果为进一步培育枝、叶中高SGAs而块茎中低SGAs的马铃薯抗性品种提供了理论依据。     

苹果PGIP基因部分家族成员启动子的克隆与功能分析

根据已发表的苹果基因组序列设计特异引物,克隆了苹果（Malus × domestica Borkh.）品种‘秦冠’和‘太平洋玫瑰’中PGIP家族基因PGIP1和PGIP2的启动子片段,序列分析结果表明PGIP1与PGIP2启动子序列相似度为71%,‘秦冠’和‘太平洋玫瑰’的PGIP1、PGIP2启动子相似度都达到了99%。两个品种PGIP1启动子中TATA-box和CAAT-box的数量明显多于PGIP2,PGIP1和PGIP2启动子分别存在茉莉酸甲酯和水杨酸响应元件,暗示PGIP1和PGIP2存在不同的抗病响应途径。构建了启动子︰︰GUS融合表达载体,通过对农杆菌介导法转化烟草叶片中的GUS活性分析,比较了不同启动子的活性。结果表明：PGIP1启动子活性在品种间差异不显著;PGIP2启动子活性在品种间差异显著,‘秦冠’是‘太平洋玫瑰’的2.37倍,可能与品种抗病性差异有关;同一品种中PGIP1启动子活性显著高于PGIP2。     

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。     

Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR)

Conditional expression of harpin_Psscauses yeast cell death that shares features of cell death pathway with harpin_Pss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpin_Pss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpin_Pssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpin_Pss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100 U ml⁻¹catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpin_Pss-mediated cell death.     

A Possible Correlation between the Blood Leukocyte Formula and the Use of Glucocorticoids as Growth Promoters in Beef Cattle

Clinical analyses designed to set welfare parameters were performed on blood drawn from the caudal vein of 14 groups of cattle (young bulls and heifers) (n = 10) from 480 to 550 kg b.w., each group representative of a different farm. The leukocyte formula exhibited a lymphocytopenia in four groups compared with the values from a control group (n = 50). This finding was related to the possible illicit use of corticoids as growth promoters in meat production. The individual plasmas tested negative by two different ELISA kits for corticosteroids, but chemical analyses by LC-MS/APCI (detection limit 0.5 ng/ml) on the pooled plasma of each of the 14 groups revealed the presence of beclomethasone and fluocinolone acetonide in 3 of the 4 suspect farms. These corticosteroids are not always efficiently screened by commercially available immunoassays. The epidemiological reliability of blood analysis as a screening test for such drugs is discussed in the light of the need for quality certification of the whole meat production processes from farm to fork, and for enhanced animal welfare.     

家蚕促产卵剂的再探讨

本文就促排三号、医用催产素对促进家蚕蛾产卵的效果及试用方法进行探讨。试验证明，前者可提高产出卵经约５％，但因品种而有差异。后者对临产蛾有加速速度的作用。     

绵羊CCL19基因启动子活性分析及其转录调控元件筛选

【目的】 鉴定绵羊趋化因子C-C基序配体19(C-C motif chemokine ligand 19,CCL19)基因启动子的核心启动子区域和关键转录因子,探究该基因在转录调控方面的作用机制。【方法】 选取绵羊CCL19基因5'-侧翼序列1 000 bp,PCR扩增启动子的7个不同长度的截短片段,并连接至pGL3-Basic质粒;将重组质粒与pRL-TK质粒共转染到293T细胞中,结合双荧光素酶报告基因检测系统分析不同截短片段的相对荧光活性。利用在线预测软件分析和筛选CCL19基因核心启动子区域内的转录因子结合位点。采用定点突变技术构建转录因子结合位点缺失的荧光素酶报告载体,与pRL-TK质粒共转染到293T细胞,分析转录因子结合位点缺失质粒的相对荧光活性。【结果】 成功构建了7个不同长度(pGL3-P、pGL3-P1、pGL3-P2、pGL3-P3、pGL3-P4、pGL3-P5及pGL3-P6)的CCL19基因启动子片段的荧光素酶报告载体;采用双荧光素酶报告基因检测系统鉴定出转录起始位点上游－256/－186 bp为CCL19基因启动子核心启动子区域,表明该区域对CCL19基因转录调控有重要作用。生物信息学分析预测到该区域存在POU5F1(-201/-189 bp)、ZBTB26(-228/-217 bp)、FOXI1(-239/-228 bp)、GLI2(-255/-243 bp)和SP2(-219/-211 bp) 5个转录因子的结合位点,并成功构建了转录因子结合位点缺失的荧光素酶报告载体。双荧光素酶报告基因检测系统分析显示,POU5F1转录因子的结合位点缺失后绵羊CCL19基因转录活性极显著降低(P<0.01),FOXI1、ZBTB26、SP2转录因子结合位点缺失后绵羊CCL19基因转录活性均极显著升高(P<0.01)。【结论】 试验成功构建CCL19基因启动子荧光素酶报告载体,确定CCL19基因启动子的核心启动子区域为转录起始位点上游－256/－186 bp,并鉴定出转录因子POU5F1结合位点可能是CCL19基因转录的重要调控位点,为下一步研究绵羊CCL19基因在先天性免疫、适应性免疫和淋巴细胞迁移等方面的功能提供理论基础。