美洲拟鲽抗冻蛋白基因（afp）导入番茄的研究

将整合在Ｔｉ质粒上的ａｆｐ基因供体ＤＮＡ,用花粉管通道和子房注射方法导入番 茄品种“中蔬四号”中,对Ｄ１、Ｄ２代进行Ｓｏｕｔｈｅｒｎ杂交获得杂交带;田间抗寒性试验表明,在春季平均气温低于正常年份４．４℃条件下,转基因组植株生长势优于对照;致死温度也比对照降低２℃、说明ａｆｐ基因已整合转化番茄基因组并获得表达。     

植物低温保护剂对番茄幼苗抗寒力的影响

采用由我们研制的植物低温保护剂对番茄幼苗抗寒力的影响。用植物低温保护剂处理番茄幼苗,超氧物歧化酶（ＳＯＤ）、过氧化物酶（ＰＯＤ）、过氧比氢酶（ＣＡＴ）活性升高,丙二醛（ＭＤＡ）含量及电导率降低。脯氨酸、可溶性总糖和叶绿素含量增加;根和叶的ＴＴＣ还原率同步增大,电导率同步降低;抗寒力鉴定结果表明：番茄幼苗可抵抗－２℃─－５℃［－２℃（５ｈ）─－３℃（３ｈ）－─４℃（３ｈ）─－５℃（１ｈ）］长达１２小时的低温,田间结果表明,番茄幼苗可抵抗－２℃─４℃低温长达一周。     

转hDAF基因小鼠的构建(英文)

本工作构建了含有ｈＤＡＦ基因的转基因小鼠,以便研究ｈＤＡＦ基因能否消除异种器官移植中的排斥反应。 采用ＤＮＡ重组的方法构建ｈＤＡＦ基因的表达载体ｐＳＰ６４ＨＰ（Ｆｉｇ．１）。通过受精卵显微注射,将其中的目的基因片段,转移到小鼠体内,建立转基因小鼠。再通过Ｄｏｔ ｂｌｏｔｔｉｎｇ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ杂交方法对出生小鼠的基因组特征进行查证。 连续两次对直接裂解菌液做ＰＣＲ扩增,筛选出重组质粒（Ｆｉｇ．２＆３）,酶切图谱（Ｆｉｇ．４）和Ｓｏｕｔｈｅｒｎ杂交（Ｆｉｇ．５）分析结果与预期吻合,出现预期条带;小鼠受精卵注射后存活比率为７７．９％,受精卵的发育率为３．４％,出生小鼠中,１０．５％出现清晰杂交信号。 表明：ｈＤＡＦ基因表达载体构建成功;并整合入小鼠基因组中。     

系统感染TMV的番茄叶胞外β-1,3-葡聚糖酶

系统感染ＴＭＶ （ｔｏｂａｃｃｏ ｍ ｏｓａｉｃ ｖｉｒｕｓ）的番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．）叶胞外蛋白提取液经冰冻干燥浓缩、－ ２０℃丙酮沉淀、ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析和Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶层析纯化,获得ＰＡＧＥ均一的β－１,３－葡聚糖酶．ＳＤＳ－ＰＡＧＥ证明,它包含分子量为３６ ｋＤ 和２７ ｋＤ的两个同工酶．以昆布多糖为底物,酶的最适ｐＨ 在４．８—５．２之间,在ｐＨ ４—８稳定;酶的最适温度在３０—４０℃之间,在４０℃保温１ｈ 后酶活性不变;Ｋｍ 值为９．２ ｍ ｇ／ｍ Ｌ．在系统感染ＴＭＶ 的番茄叶胞外蛋白提取液中,有分子量为２２ ｋＤ、２７ ｋＤ和３６ ｋＤ的３个β－１,３－葡聚糖酶同工酶     

异戊烯基转移酶基因转化胡卢巴的研究

将含有生长素诱导的小分子ＲＮＡ启动子与异戊烯基转移酶基因的融合基因（ｐＳＡＵＲ－ｉｐｔ）的Ｔｉ质粒作为供体ＤＮＡ,通过花粉管通道法导入胡巴中。Ｄ１、Ｄ２代植株经Ｓｏｕｔｈｅｒｎ杂交检测,有杂交带出现,获得了转基因胡卢巴。对转基因胡卢巴Ｄ３、Ｄ４和Ｄ５代进行了生理生化指标及田间性状分析,发现转基因胡卢巴与ＣＫ相比有植株略矮、分枝数增多、双角数增多等表型变化,Ｄ４代叶绿素含量、半乳甘露聚糖含量增加,Ｄ     

HIV21 gag基因和gp120基因转化番茄及转基因植株再生

构建了HIV 1的gag基因、gp1 2 0基因及gag gp1 2 0嵌合基因的植物双元表达载体。通过农杆菌介导法将HIV 1的gag基因、gp1 2 0基因和gag gp1 2 0嵌合基因导入番茄 ,获得抗性转化再生植株。PCR检测和Southern杂交鉴定目的基因已整合到再生植株基因组中 ,获得了转基因植株。GUS染色及Northern杂交结果表明目的基因已得到表达。本实验首次进行了HIV 1抗原基因转化番茄的研究 ,为利用番茄生产艾滋病新型口服疫苗打下了良好的基础。     

人DAF基因在转基因小鼠中的遗传与表达

为研究人ＤＡＦ基因在小鼠体内遗传与表达的规律,从质粒ｐＳＦＦＶ－ＤＡＦ分离出一段包含人ＤＡＦ基因的ＤＮＡ片段。采用受精卵显微注射法建立转人ＤＡＦ基因小鼠。提取出生小鼠的染色体ＤＮＡ,经Ｄｏｔ－ｂｌｏｔ与Ｓｏｕｔｈｅｒｎ－ｂｌｏｔ杂交相结合确定首建转基因小鼠,并经Ｄｏｔ－ｂｌｏｔ杂交研究人ＤＡＦ基因在转基因小鼠体内的遗传特征,Ｎｏｒｔｈｅｒｎ杂交确定其表达情况。小鼠受精卵经基因导入后,共生出２４只小鼠,其中４只被确定为首建转基因小鼠,整合率为１５％,在首建转基因小鼠两两交配生出的Ｆ１代小鼠中分别有７０％和７５％继续携带人ＤＡＦ基因。首建转基因小鼠中有１只小鼠在ＲＮＡ水平表达了人ＤＡＦ基因。可见,人ＤＡＦ基因整合入小鼠基因组中,并能够稳定遗传及表达。     

两种AFP阳性肝癌细胞专一基因表达调控序列的构建

构建了由人胰蛋白酶抑制基因的增强子和大鼠ａｆｐ基因启动子与沉默子组成的杂合基因表达调控序列;还构建了１．２ｋｂ的由大鼠ａｆｐ基因增强Ⅲ和ｒＰＳ组成的调控序列。将报告基因－氯霉素乙酰转移酶基因置于这些序列控制之下,构建成ＣＡＴ表达载体ＡＴｒＰＳ－ｐＣＡＴ和ｒＡＦＰ－ｐＣＡＴ。     

通过农杆菌介导将番茄铁转运蛋白基因导入八棱海棠

用农杆菌介导法,成功地将番茄铁转运蛋白基因导入了苹果砧木八棱海棠.获得了19个卡那霉素抗性株系,其中有11个株系经PCR鉴定为阳性.Southern杂交结果显示:有9个转基因株系基因组中整合了完整的目的基因.选择其中含有单拷贝和3个拷贝目的基因的各一个株系进行水培试验,结果表明整合了单拷贝目的基因的转基因株系表现出较强的抗缺铁胁迫能力,5周后其植株的鲜重比对照高21%～34%.     

原位杂交及原位PCR检测幽门螺杆菌

本研究建立了检测幽门螺杆菌的原位杂交和原位ＰＣＲ技术,采用ＰＣＲ掺入的方法标记原位杂交的生物素探针,６份ＨＰ阳性胃活检组织冰冻切片杂交均为阳性,６份阴性对照组织为阴性。９／１２份ＨＰ阳性对照组织石蜡切片杂交阳性,２份阴性对照切片为阴性。３份阳性对照猫胃粘膜冰冻切片杂交也是阳性。原位ＰＣＲ的引物来自ＨＰ的１６ｓｒＲＮＡ基因,在扩增过程掺入生物素基因。４／４例ＨＰ阳性人胃粘膜冰冻切片原位扩增阳性,２／     

抗冻蛋白(afp)基因表达载体的构建及对香蕉胚性细胞悬浮系的转化

通过PCR从‘京都七寸人参'胡萝卜基因组DNA中扩增抗冻蛋白基因,测序结果表明该基因的核苷酸序列与从宁夏‘吴忠'胡萝卜中克隆的完全一致。先后将获得的胡萝卜afp基因克隆和亚克隆至pMD18-T和pBI121,构建植物表达载体pBI121-afp。通过冻融法将pBI121-afp导入根癌农杆菌EHA105中。以香蕉栽培品种‘北大矮蕉'的胚性细胞悬浮系为受体,采用农杆菌介导法将胡萝卜afp基因导入其中,然后在Kanamycin的选择压力下通过体细胞胚发生途径进行植株再生。共获得抗性再生植株9株,其中两株经PCR检测呈阳性,可初步确定目的基因已经整合到这两株转基因香蕉植株的基因组中。     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its     

抗虫基因转化优良籼型保持系D297B的研究

本文报道了用基因枪法将雪花莲外源凝集素基因(gna)转移到优良籼型杂交稻保持系D297B中。通过PCR、Southern blotting和Western blotting等分子检测方法证明,外源基因已整合到受体材料的基因组中并得到表达。蛋白活性测定结果显示转基因在受体中表达的蛋白具有生物活性。潮霉素抗性分析表明外源基因多数是以单位点形式整合并以3：1方式遗传。另外,PCR证据也说明抗虫基因与选择标记基因基本上是共整合和共遗传的。     

抗虫基因转化优良籼型保持系D297B的研究

本文报道了用基因枪法将雪花莲外源凝集素基因(gna)转移到优良籼型杂交稻保持系D297B中。通过PCR、Southern blotting和Western blotting等分子检测方法证明,外源基因已整合到受体材料的基因组中并得到表达。蛋白活性测定结果显示转基因在受体中表达的蛋白具有生物活性。潮霉素抗性分析表明外源基因多数是以单位点形式整合并以3∶1方式遗传。另外,PCR证据也说明抗虫基因与选择标记基因基本上是共整合和共遗传的     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.     

抗冷冻蛋白基因(afp)转化大豆的研究

目的:通过农杆菌介导法遗传转化大豆。方法:通过热激法将质粒pCAAFP66导入根癌农杆菌菌株EHA105中获得含有抗冷冻蛋白基因(afp)及除草剂抗性筛选标记基因(bar)的农杆菌工程菌株;以大豆品种华春6号和马祖1号种子的下胚轴为外植体,经过农杆菌介导将抗冷冻蛋白基因导入大豆基因组中,在含有除草剂草丁膦(PPT)的培养基中筛选、并经过PCR鉴定获得大豆转化植株。结果:PPT的最佳筛选浓度为1.0mg/L,华春6号和马祖1号的阳性植株数分别为6株和2株,转化效率分别为3.70%和0.94%。结论:不同基因型大豆的转化率存在差异,抗冷冻蛋白基因成功遗传转化进大豆细胞中。     

一种检测转1Dx5基因小麦植株的新方法

目的:建立一种有效区分Glu-1D等位基因的Multiplex-PCR体系,快速检测转1Dx5小麦植株。方法:根据1Dx5和1Dx2亚基基因的区别设计特异的PCR引物,通过多重PCR技术检测花粉管通道法转化1Dx5核心片段的转基因T1代植株。结果:Multiplex-PCR能够在转基因T1代材料中扩增Glu-1D等位基因的特征条带,分别为343bp、320bp的1Dx5基因特异片段以及361bp的1Dx2基因特异片段,与预期结果一致。结论:该技术能够检测多个靶基因,有效地区分转基因Glu-1D上的等位基因,证实外源1Dx5基因已整合到受体基因组中,对检测基因组庞大、外源基因序列GC含量高且与内源基因同源性高的转基因小麦十分有效。