Ri质粒介导TMV和CMV外壳蛋白基因转化甜椒研究

利用发根农杆菌（Ａｇｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ）的Ｒｉ质粒介导的二元载体,将ＰＢＴＣ－８质粒上的ＴＭＶ－ｃｐ和ＣＭＶ－ｃｐ基因导入甜椒中,用共培养的方法,诱导愈伤组织,经卡那霉素的抗性筛选,进一步的培养获得再生植株,经ＰＣＲ和ＥＬＩＳＡ的检测呈阳性反应,转化植株田间人工接种为抗性,证明ＴＭＶ－ｃｐ和ＣＭＶ－ｃｐ基因导入甜椒植株并已表达。     

VAM真菌对再植桃实生苗矿质元素吸收的影响

着重分析了土壤灭菌和接种ＶＡＭ（ｖｅｓｉｃｕｌａｒ－ａｒｂｕｓｃｕｌａｒ ｍｙｃｏｒｒｈｉｚａ）真菌对再植桃实生苗矿质元素吸收的作用。研究表明土壤灭菌后接种ＶＡＭ真菌Ｇｌｏｍｕｓｅｐｉｇａｓｅｕｍ、Ｇ．ｍｏｓｓｅａｅ、Ｇ．ｍａｃｒｏｃａｒｐｕｓ，能促进桃实生苗对Ｐ、Ｂ、Ｃａ、Ｋ、Ｍｇ、Ｍｎ的吸收，对于克服由营养元素缺乏引起的桃连作障碍有较大潜力。但在同一土壤处理中，各菌种表现不同；同一菌种对各元素的作用也不同。     

山杏等砧木对仁用龙王帽杏生育状况的影响

关于苹果、葡萄等果树砧木的研究较多。关于杏树砧木的研究报道很少,有的报道指出,山杏［Ａ．ｖｕｌｇａｒｉｓＬａｍ．ｖａｒ．ａｎｓｕ（Ｍａｘｉｍ）］、西伯利亚杏（Ａ．ｓｉｂｉｒｉｃａＬａｍ．Ｖａｒ．ｓｉｂｉｒｉｃａ）和毛桃（Ａ．ｐｅｒｓｉｃａＬ．ｖａｒ．...     

西瓜花叶病毒2号外壳蛋白基因克隆及其在大肠杆菌中的表达

以郑州地区西瓜花叶病病叶分离物──西瓜花叶病毒２号（２－ＷＭＶ－）为毒源，从繁殖寄主西葫芦病叶中分离、鉴定了病毒。以病毒基因组ＲＮＡ为模板，逆转录合成ｃＤＮＡ，通过多聚酶链式反应（ＰＣＲ），从ｃＤＮＡ中扩增和克隆了病毒外壳蛋白（ＣＰ）基因，其中包括３’端非编码区，完成了全部核苷酸序列分析。ＣＰ基因全长１０９９个苷耷酸，其中编码区长９０３个核苷酸，编码３０１个氨基酸，３’端非编码区长１９６个核苷酸。Ｚ－ＷＭＶ－２ＣＰ基因的编码区比Ａ－ＷＭＶ－２，Ｕ－ＷＭＶ－２两株系的ＣＰ基因编码区分别长出６０个核耷酸，多编码２０个氨基酸。与两个株系相比，编码区的核昔酸序列同源性分别为８８．５％和８７．０％，氨基酸序列同源性分别为９０．０％和８８．７％，３，端非编码区的同源性分别为６８．３％和７１．８％。若不考虑Ｚ－ＷＭＶ－２多出的６０个核苷酸及２０个氨基酸，则上述同源性依次为９５．４％、９３．７％、９６．４％、９５．０％、８８．８％和９３．４％。构建了含ＷＭＶ－２ＣＰ基因的大肠杆菌表达载体，经聚丙烯酰胺凝胶电泳分析，表达出了与天然病毒外壳蛋白分子量相同的蛋白。     

苦瓜抗虫性研究进展

苦瓜的抗虫性很强。美国、日本、印度等国家对苦瓜的抗虫表现及抗虫机理进行了较深入的研究。１．抗虫表现　苦瓜对黄守瓜、西葫芦红守瓜（Ａｕｌａｃｏｐｈｏｒａｆｏｖｅｉｃｏｌｌｉｓ），（ＣｈａｎｄｒａｖａｎａＭ．Ｖ．１９８７）、Ｓｐｏｄｏｐｔｅｒａｌｉｔｕｒａ、东方粘虫（Ｐｓｅｕｄａｌｅｔｉａｓｅｐａｒａｔａ）（ＹａｓｕｉＨ．ＫａｔｏＡ．ｅｔａｌ．１９９８）、Ａｔｈａｌｉｓｐｒｏｘｉｍａ（ＫｕｍａｒＡ．１９７９）具有抗性。苦瓜对这些昆虫的抗性程度不一样。ＹａｓｕｉＨ．等（１９９８）报道，苦瓜对东方粘虫的…     

加州大学戴维斯分校芹菜育种

加州大学戴维斯分校芹菜育种武峻新（译）芹菜（ＡｐｉｕｍｇｒａｖｅｏＬｅｎｓＬ．）在美国加州是一种重要蔬菜。在加州Ｖｅｎｔｕｒａ、Ｍｏｎｅｒｅｙ、ＳａｎＬｕｉｓＯｂｉｓｐｏ和ＳａｎｔａＢａｒｂａｒａ县的沿海地区大规模种植。根据１９８９年统计，当年共收获...     

提高苹果基因转化效率的研究

采用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ）强株系ＥＨＡ１０５（ｐ３５ｓＧＵＳ－ｉｎｔｒｏｎ）研究了影响‘皇家嘎拉’苹果（Ｍａｌｕｓ ｄｏｍｅｓｔｉｃａ Ｂｏｒｋｈ．）外植体的β－葡萄糖醛酸酶（ＧＵＳ）基因的瞬时、稳定表达水科和转基因植株的再生。证明在培养基生长素（ＩＢＡ、ＮＡＡ）存在的条件下，外植体的ＧＵＳ基因的瞬时表达水平提高了３～４倍，而共培养两周后稳定表达水平提高２     

芒果果实冷害过程中谷胱甘肽和抗坏血酸含量的变化

‘紫花’芒果果实（ＭａｎｇｉｆｅｒａｉｎｄｉｃａＬ．ＺｈｉＨｕａ）２℃贮藏１５ｄ时发生严重的冷害。贮藏前期,还原型谷胱甘肽（ＧＳＨ）、还原型维生素Ｃ（ＡｓＡ）含量略有上升,说明短期低温胁迫可促使机体提高清除氧自由基能力,以适应不良环境。随冷害时间延长,ＧＳＨ、ＡｓＡ含量下降;膜脂过氧化产物（ＭＤＡ）、膜透性、氧化型谷胱甘肽（ＧＳＳＧ）和氧化型维生素Ｃ（ＤＨＡ）增加,表明细胞防御能力下降,自由基代谢平衡可能被打破,膜脂过氧化加剧,果实冷害加重。１０℃贮藏的芒果,无冷害症状,２２ｄ时果实启动成熟,贮藏期间ＧＳＨ、ＡｓＡ含量维持较高水平。     

葡萄属植物核糖体基因的RFLP分析

葡萄属植物核糖体基因的ＲＦＬＰ分析张立平１林伯年２沈德绪２董继新２（１浙江农业大学生物科学系；２浙江农业大学园艺系，杭州３１００２９）关键词葡萄属；核糖体基因；ＲＦＬＰＡｎａｌｙｓｉｓｏｆＲＦＬＰｉｎＲｉｂｏｓｏｍａｌＤＮＡｏｆＶｉｔｉｓＺｈａｎｇＬ...     

大白菜核基因互作雄性不育系91—5A遗传机制初探

大白菜核基因互作雄性不育系９１—５Ａ遗传机制初探张书芳，赵雪云，周邦福（沈阳市农业科学院，１１００３４）关键词核基因互作，显性不育基因，显性上位可育基因ＲｅｓｅａｒｃｈｏｎＧｅｎｅｔｉｃＭｅｃｈａｎｉｓｍｏｆＩｎｔｅｒａｃｔｉｖｅＧｅｎｉｅＭａｌｅＳ...     

柑橘衰退病晚期抑制差减cDNA文库的构建及初步分析

为了研究柑橘衰退病晚期应答的分子机理,利用经典的抑制差减杂交技术,以感染柑橘衰退病3年后的‘不知火’杂柑实生苗叶片组织的c DNA为测试方,以脱毒的‘不知火’实生苗叶片组织的c DNA为驱动方进行差减杂交,构建了柑橘衰退病诱导的正向抑制差减c DNA文库。随机挑取此文库中210个阳性克隆进行测序,获得了192条有效序列。将这些EST序列聚类拼接后,共获得77条非重复序列。对这些EST进行GO和KEGG分析,发现这些EST的功能主要与胁迫及防御、代谢、转录、转运、蛋白命运等途径相关。用实时定量RT-PCR验证了其中4个基因:几丁质酶基因、茉莉酸响应基因的转录抑制因子1基因(JAZ1)、钙调素结合蛋白基因、咖啡酸—氧甲基转移酶基因,结果表明在衰退病的诱导下,‘不知火’杂柑叶片中这4个基因的表达量均有明显提高,说明这些基因可能参与了‘不知火’杂柑叶片晚期衰退病应答。JAZ1基因的上调表达表明可能宿主衰退病晚期应答过程中的诱导系统抗性受到了抑制。     

Anti-adenovirus effect of cinnamaldehyde

AIM: To investigate the anti-adenovirus effect of cinnamaldehyde in vitro. METHODS: The anti-adenovirus effect of cinnamaldehyde was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and virus proliferation inhibition experiment. The expression of hexon protein was measured by immunohistochemistry. RESULTS: The results of MTT and virus proliferation inhibition experiment showed that cinnamaldehyde enhanced the survival rate of the host cells, and decreased adenovirus titer by the ways of direct virus inactivation, anti-adenovirus in the stage of biosynthesis and adsorption of the adenovirus. The viability of the host cells had a positive correlation with the drug concentration under the condition of virus infection. Cinnamaldehyde did not block the virus uptake into host cells, and did not protect the host cells from infection. The expression of hexon protein was significantly lower (P<0.05) in cinnamaldehyde group than that in virus group.CONCLUSION: Cinnamaldehyde has anti-adenovirus effect, but can not directly protect host cells. The anti-adenovirus effect of cinnamaldehyde may be related with the inhibition of hexon protein expression.     

Effect of respiratory syncytial virus on apoptosis and expressions of FasL,Fas, Bcl-2 and Bax

AIM: To study the relationship between respiratory syncytial virus (RSV) infection and apoptosis, between RSV infection and expressions of FasL, Fas, Bcl-2 and Bax. METHODS: Apoptotic cells were examined by flow cytometry and transmission electron microscope. Immunohistochemical analysis was used to detect the expressions of apoptosis-associated gene FasL, Fas, Bcl-2 and Bax in A549 cells during RSV infection. RESULTS: Apoptotic index increased at 72 h and 120 h postinfection. Apoptotic cells were detected by transmission electron microscope. High-expressions of FasL, Fas and Bax genes and low-expression of Bcl-2 gene were detected by immunohistochemical staining. CONCLUSION: Apoptosis in A549 cells was induced by RSV infection. This apoptosis may be induced by up-regulating the expression of FasL, Fas, Bax genes and down-regulating the expression of Bcl-2 gene.     

莲藕根状茎膨大相关基因的挖掘与表达分析

 研究发现，莲藕根状茎干物质、可溶性糖及可溶性蛋白含量随着根状茎发育而逐步增加，尤其在根状茎发育后期含量增加更加明显。在此基础之上，利用转录组测序技术，归纳并分析 86个可能与莲藕根状茎膨大相关基因，分别为35个激素诱导蛋白基因、4个光诱导蛋白（MADS-BOX）基因、11个根状茎贮藏蛋白基因（Patatin）、35个与淀粉代谢相关基因以及1个与根状茎形成相关基因。数字基因差异表达谱研究结果表明，根状茎贮藏蛋白家族基因（Lrplp6、Lrplp9、Lrpfp1、Lrprp、Lrpfp2、Lrplp4、Lrplp5、Lrplp8、Lrpp、Lrplp2）和淀粉合成相关基因（Lrgbss、Lrsbe1、Lrsbe2、LrsbeII、LrsbeIII）在根状茎形成后期表达量高，表达丰度均为初期的3倍以上，而其他基因变化相对较小。半定量RT-PCR结果进一步证明上述数字基因差异表达谱结果的可靠性，同时也表明Lrplp8和Lrgbss的表达与根状茎膨大具有高度相关性。由此认为，上述10个贮藏蛋白合成相关基因和5个淀粉合成相关基因，尤其Lrplp8和Lrgbss，对莲藕根状茎的膨大起到重要作用。     

葡萄A病毒四川分离物的外壳蛋白基因克隆与原核表达

葡萄A病毒（Grapevine Virus A,GVA）是葡萄病毒属（Vitivirus）的典型种,在世界葡萄产区广泛分布。采集10株“藤稔”葡萄成熟枝条,使用6种葡萄病毒ELISA试剂盒检测发现10个样本中有6个感染4种不同的葡萄病毒。以GVA的ELISA阳性植株为材料进行RT-PCR扩增,首次获得了GVA四川分离物SL10的完整外壳蛋白基因（CP）。该基因全长597 bp,将其与GeneBank收录的15个GVA分离物的CP序列进行比对和构建系统进化树。把不同地理起源的GVA分离物分成2个变异组;其中Ⅰ组包括3个分离物(与Ⅱ组的其他分离物只有75.9%～80.1%的序列同一性);其余的13个分离物组成Ⅱ组（组内分离物具有84.4%～99.5%的序列同一性）。构建了GVA CP的原核表达质粒PET-30-GVAcp并转化BL21菌株,经IPTG诱导,目的基因得到了大量表达。     

西瓜抗病毒RNAi植物表达载体的构建

为了研究转化同一种病毒的不同基因在转基因西瓜中引发RNA沉默的效果以及利用RNA沉默培育抗多种病毒的策略,以pFGC5941为骨架质粒,分别构建了ZYMV cp、nib和Hc-Pro基因的反向重复植物表达载体pFIRZYMVCP、pFIRZYMVNIb和pFIRZYMVHc-Pro;利用重叠延伸PCR的方法,首先构建了含有CMV cp、WMV cp和ZYMV cp部分基因片段的CWZ cp三价基因,采用该三价基因构建了CWZ cp基因的反向重复植物表达载体pFIRCWZ CP。研究首次在国际上构建了西瓜转基因抗病毒RNAi植物表达载体,为探讨RNA沉默在转基因抗病毒西瓜中的应用奠定了基础。     

钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选

为了筛选适合于钝裂银莲花类黄酮/花青素合成途径中相关基因qRT-PCR表达分析时的内参基因,根据钝裂银莲花蓝/白不同花色花器官组织的转录组测序结果,选取了多聚泛素酶基因(polyubiquitin,UBQ)、微管蛋白基因(β-tubulin,-TUB)、水通道蛋白基因(aquaporin,AQP)、肌动蛋白基因(act...     

Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N. I- Ceu I/PI- Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5'040.pkGFP-II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'.040.CMV.RFP-N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5'.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein (EGFP). RESULTS: The recombinant adenovirus vector AdH5'.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×10¹⁵ vp/L and 1×10¹³ pfu/L,respectively. The infection efficiencies of recombinant adenovirus vector Ad5'.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP (P<0.05). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.     

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.     

Microarray analysis of tumor metastasis-related gene expression in PSMA-silencing prostate cancer cells

AIM: To investigate the functions of prostate-specific membrane antigen (PSMA) in prostate cancer metastasis. METHODS: Specific siRNA to knock down PSMA expression was designed and transfected into LNCaP cells. The tumor metastasis gene chip was also used to analyze the differential expression of 84 genes related to cancer metastasis. RESULTS: Specific siRNA was successfully designed and constructed and the gene expression of PSMA in LNCaP cells was knocked down. The RNAi efficiency was more than 75% at mRNA level and more than 68% at protein level. The results of the tumor metastasis gene chip indicated that 10 genes were up-regulated (such as CDH6 and CXCL12) and 4 genes were down-regulated (such as CCL7 and MDM2) in the LNCaP cells treated with PSMA siRNA. CONCLUSION: The PSMA is involved in the regulatory pathways in prostate cancer metastasis.