果蔬采后炭疽病病原菌的分子识别

本研究旨在分析果蔬采后炭疽病痛原菌菌种特异的分子特征,为建立早期分子检测方法奠定基础.利用筛选的炭疽病病害标本,通过对微观特征观察及柯贺氏法则的验证,确定5种病原菌作为研究对象.采用真菌核糖体基因转录间隔区域(ITS)通用引物进行PCR扩增,在序列分析的基础上,设计特异性引物.首次为多种疽病菌的准确鉴定和采后炭疽病的快速、准确的检测提供了技术和方法.     

Evaluation of cryodamage in small abalone,Haliotis diversicolor,spermatozoa by using a flow cytometer

We used flow cytometry to determine the quality of small abalone, Haliotis diversicolor, sperm before freezing and after thawing. We investigated the effects of cryopreservation on the characteristics of small abalone semen and determined the motility and fertilization capacity of the pre-freezing and post-thawed sperm. The percentages of motility and fertility were 61 ± 2% and 67 ± 1%, respectively, for the post-thawed sperm and 90 ± 4% and 92 ± 0%, respectively, for the pre-freezing sperm. Sperm cells were stained with specific fluorescent dyes to measure plasma and acrosomal membrane integrity, mitochondrial status, oxidation level, DNA compaction, and viability through flow cytometry. The frozen–thawed sperm exhibited significantly higher mitopotential activity (p < 0.05, damaged mitochondria; 25.01 ± 1.18%) and oxidation value (p < 0.01, free radicals; 63.79 ± 3.93%) compared with the pre-freezing sperm. The oxidation level was the most sensitive signal of the cryopreservation-induced small abalone sperm damage. Flow cytometry is valuable for the objective, accurate, and rapid assessment of pre-freezing and post-thaw small abalone sperm quality.     

"壮阳促孕散"对小鼠雌激素样作用观察

采用常规药理实验方法,探讨中药壮阳促孕散对雌性幼小鼠子宫增重及血清雌二醇含量的影响.结果表明:壮阳促孕散能使幼年小鼠子宫显著增重,血清雌二醇含量明显增高,从而证明中药壮阳促孕散具有雌激素样作用.     

用作植物疫苗的转基因胡萝卜的分子生物学筛选与鉴定

以包含有不同外源病毒基因的转基因胡萝卜为试验材料,应用分子生物学手段,从DNA水平上和蛋白质水平上对这些材料加以鉴定,经PCR引物筛选后得到转基因胡萝卜的阳性率为38%左右;为保证实验的可靠性和可行性,防止检测过程中遗漏和错误的发生,用特异的PCR引物对检测结果进行复选,得到的植株阳性率为7%.在蛋白水平上应用ELISA检测法检测获得的阳性植株的结果与PCR检测结果相同,且同时发现外源基因在胡萝卜根中的表达量明显小于在叶中的表达量.     

Establishment of the Method of Immunohistochemistry Assay for the Detection of Scrapie in Chinese Short-Tailed Han Sheep by Monoclonal Antibody

The method of immunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep was established using monoclonal antibody. Genomic DNA was isolated from Chinese Short-tailed Hart sheep blood. Using the polymerase chain reaction technique, PrP27-30 gene sequence was amplified from Chinese Short-tailed Han sheep genomic DNA. By recombinant DNA technology, the recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained. Then, using standard methodology of myeloma cell fusion, a panel of monoclonal antibodies was generated. With mAbs, scrapie in Chinese Short-tailed Han sheep was detected by immunohistochemistry assay. The recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained and a panel of six hybridoma cell lines secreting specific antibodies to Chinese Short-tailed Han sheep PrP27-30 related to scrapie was obtained with one fusion between myeloma Sp2/0 and spleen ceils from mice immunized with the purified recombinant protein. Four hybridoma cell lines can be used in immunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep. So that the special monoclonal antibody developed in author＇s institute can be used to detect PrP^sc of scrapie in Chinese Short-tailed Han sheep by immunohistochemistry in China.     

鸡CaBP-D28k基因重组质粒构建

    为构建pCEP4-CaBP-D28k真核表达载体,根据GenBank已发表钙结合蛋白D28k(calbindin-D28k,CaBP-D28k)设计1对引物,利用RT-PCR方法获得新扬州鸡CaBP-D28k基因序列,克隆入真核表达载体pCEP4,经PEI介导转入非洲绿猴转化肾细胞(COS-1)中,并以间接免疫荧光试验(IFA)检测CaBP-D28k表达状况.结果表明,CaBP-D28k基因核苷酸长度为789 bp,编码261个氨基酸,与GenBank上已发表序列比较,核苷酸同源性为99.9%,在第760位处存在G→T的错义突变.该序列与蟾蜍、鼠、人CaBP-D28k氨基酸同源性分别为79.7%、78.2%、78.6%;酶切和测序鉴定证实pCEP4-CaBP-D28k含有目的片段,且连接构建正确;在COS-1细胞中检测到CaBP-D28k表达.这为进一步研究pCEP4-CaBP-D28k在动物体中的表达,调控机体钙代谢奠定了基础.     

玻璃化冷冻保存对体外成熟猪卵母细胞DNA的影响

采用乙二醇(EG) 二甲基亚砜(DMSO)作为冷冻保护剂处理并使用微管法冷冻保存猪卵母细胞,运用细胞形态观察和单细胞凝胶电泳(SCGE)方法检测玻璃化冷冻过程对细胞DNA损伤情况.结果显示:保护剂处理冷冻组,细胞形态正常率和DNA异常率分别为63.364%、42.059%;同保护剂处理未冷冻对照组82.571%、25.758%的差异显著(P<0.05).表明冷冻保存过程对猪卵母细胞DNA有一定影响:以无冷冻保护剂处理卵母细胞为对照检测保护荆的细胞毒性,对照组形态正常率和DNA异常率分别为84.51%、12.28%,与冷冻保护剂处理组有显著差异(P,0.05).说明冷冻保护荆对猪卵母细胞DNA有明显细胞毒性作用.     

湖南省猪瘟流行情况的血清学调查

采用ELISA对湖南省规模猪场送检的953份血清进行了猪瘟抗体的检测,对305个病例（场次）进行了猪瘟抗原的检测。结果表明未免疫猪的抗体阳性率仅为2%,免疫猪的抗体阳性率为55.59%;305个病例（场次）其猪瘟抗原的阳性率为61.97%。     

葡萄逆境胁迫诱导启动子的克隆及表达分析

为了研究葡萄抗逆基因(CAN70200.1)的表达特性,采用PCR技术从葡萄中克隆了CAN70200.1基因上游一段长度为1 354 bp的启动子片段,命名为PCAN。采用Plant CARE和PLACE启动子在线预测工具分析表明,PCAN启动子序列具有CAAT-box、TATA-box基本的顺式作用元件和一些参与非生物胁迫、光和植物激素应答相关的顺式作用元件。为验证启动子的表达特性,将PCAN启动子连接到p CAMBIA1391Z载体GUS基因的上游,构建成植物表达载体p1391Z-CAN,并通过农杆菌介导法转化烟草,经PCR鉴定,获得转基因植株。对转基因烟草植株进行逆境胁迫处理发现,在干旱处理120 min后,PCAN启动子活性达到最强;而4℃低温处理30~60 min时,PCAN启动子活性达到最强,表明PCAN启动子具有低温和干旱胁迫诱导表达特性。