外源抗药性基因导入香菇体内的研究

利用PEG转化法将携有潮霉素抗药性基因的质粒载体导入香菇原生质体内，获得了抗性转化子，但转化率较低，每微克DNA仅有0．5个转化子。点杂交证实，外源基因已整合到香菇染色体；不同转化子在抗笥平板上生长速率不同，且转化子有丝分裂是稳定的。香菇转基因技术的成功，为利用该技术克隆香菇内源基因及启动子，将外源有益目的基因导入香菇体内，为培育香菇基因工程菌株提供了可能。     

离子束处理将外源基因导入西瓜研究初报

探讨了借助离子束处理将银杏基因簇ＤＮＡ和耐辐射异常球菌基因簇ＤＮＡ导入西瓜并使其表达的可能性。结果表明，用离子束处理西瓜种子后，供体ＤＮＡ与西瓜受体ＤＮＡ在当代实现了超远缘分子杂交，杂交后银杏内酯在西瓜品种３－１６和ＳＲ２－ｌ４－２中的表达量分别为１７．０７５６μｇ／ｇ和４５．９９９８μｇ／ｇ，其杂交的亲和率为２５％，而ＳＯＤ在其中的表达比对照提高１倍以上的杂交亲和率为２１％。     

黑籽南瓜DNA导入西瓜后子代RAPD标记的变化

应用花粉管通道导入技术将黑籽南瓜ＤＮＡ导入受体早花西瓜，经两次传代后，得到有变异性状的子代Ｄ２－１、Ｄ２－２、Ｄ２－３，对三个子代及原始亲本黑籽南瓜和早花西瓜进行了ＲＡＰＤ分析。     

香菇线粒体DNA限制性片段长度多态性

研究了来自不同地理区域的５１个香菇野生菌株的线粒体ＤＮＡ（ｍｔＤＮＡ）的限制性片段长度多态性（ＲＦＬＰｓ），其中３８株来自日本，７株来自巴布新几内亚，４株来自新西兰，１株来自婆罗州，１株来自泰国。把它们的ｍｔＤＮＡｓ用限制酶ＥｃｏＲＩ、ＢａｍＨＩ消化，分别产生２４、１２种ＲＦＬＰ型。经比较，５１个野生香菇菌株的ｍｔＤＮＡ有２８种表型。以共迁移限制性片段为基础，计算表型对的相似矩阵，采用ＵＰＧＭＡ聚类，Ｆｉｔｃｈ－Ｍａｒｇｏｌｉａｓｈ法分析，结果同时表明香菇ｍｔＤＮＡ表型分成５群，与各自的地理区域相对应。本研究所得到的系统发育关系证实先前所作的同工酶分析结果，在明香菇存在几个种内组，它们的线粒体和核基因组存在遗传差异。     

黑籽南瓜DNA导入西瓜后子代RAPD标记的变化

应用花粉管通道导入技术将黑籽南瓜ＤＮＡ导入受体早花西瓜，经两次传代后，得到有变异性状的子代Ｄ_２－１、Ｄ_２－２、Ｄ_２－３。对三个子代及原始亲本黑籽南瓜和早花西瓜进行了ＲＡＰＤ分析。     

瓠瓜DNA导入西瓜产生抗枯萎病变异的遗传研究

采用ＤＮＡ浸胚法将瓠瓜ＤＮＡ导入西瓜，在病圃中经３代自交纯化和抗性筛选，在Ｄ３代选育出了Ｄ３－１～Ｄ３－４４份抗性材料。苗期接种鉴定结果表明，Ｄ３－１和Ｄ３－２对西瓜枯萎病高抗，Ｄ３－３和Ｄ３－４对西瓜枯萎病中抗。用感病品种蜜宝作母本，分别以高抗西瓜枯萎病的材料Ｄ３－１和Ｄ３－２作父本杂交，Ｆ１代都表现高抗枯萎病。Ｆ２代及与感病亲本蜜宝回交的ＢＣ１代群体抗、感分离比例都符合３∶１及１∶１的分离比例，说明枯萎病抗性遗传是单基因或单ＤＮＡ片段控制的显性遗传。     

分子生物学技术在菇类研究中的应用

本文报道了应用ＲＦＬＰｓ和ＲＡＰＤ技术分析香菇类缘关系，鉴别品系及分析木耳杂交后人攻原体质体融合子的结果，发现供试的３２个香菇菌株的总ＤＮＡ限制性内切酶片段长度存在多态性；木耳杂交后代、融合子及其亲株间ＤＮＡ ＰＣＲ扩增产物谱带型出现差别，这充分说明ＲＦＬＰｓ和ＲＡＰＤ技术在分析遗传变异、鉴别品系、鉴定杂种和融合子研究中的重要作用。     

外源DNA导入技术在蔬菜育种上的应用前景

外源ＤＮＡ导入技术在蔬菜育种上的应用前景韩玉珠（吉林农业大学园艺系）１９７８年周光宇等利用棉花授粉后形成的自然花粉管通道，将外源ＤＮＡ导入受体引起了棉花性状变异，首次创立了授粉后外源ＤＮＡ导入植物的技术［１，１４］。外源ＤＮＡ导入技术，是植物分子育种...     

含ipt基因生长素调控基因根癌农杆菌转化牵牛萌动种胚同工酶分析

以牵牛萌动种胚为受体，用含有３５Ｓ启动子－Ｇｕｓ基因－ｉｐｔ基因－Ｎｏｓ基因的根癌农杆菌ＬＢＡ４４０４３及含有３５Ｓ启动子－Ｇｕｓ基因－生长素调控基因－Ｎｏｓ基因的根癌农杆菌ＬＢＡ４４０４进行转化。通过对转化及对照植株苗期叶的同工酶分析，发现转化植株叶的过氧化物酶同工酶及酯酶同工酶谱带来对照植株有明显差异。转化频率较高。间接验证了外源ＤＮＡ的导入，说明同工酶分析方法可做为转基因植株早期筛选的方法之     

瓠瓜DNA导入西瓜产生抗枯萎病变异的遗传研究

采用ＤＮＡ浸胚法将瓠瓜ＤＮＡ导入西瓜,在病圃中经３代自交纯化和抗性筛选,在Ｄ３代选育出了Ｄ３－１￣Ｄ３－４ ４份抗性材料。苗期接种鉴定结果表明,Ｄ３－１和Ｄ３－２对西瓜枯萎病高抗,Ｄ３－３和Ｄ３－４对西瓜枯萎病中抗。用感病品种蜜宝作母本,分别以高抗西瓜枯萎病的材料Ｄ３－１和Ｄ３－２作父本杂交,Ｆ１代都表现高抗枯萎病。Ｆ２代及与感病亲本蜜宝回交的ＢＣ１代群体抗、感分离比例都符合３：１及１：１的分离比     

10-23 deoxyribozymes inhibit the expression of bacterial β-lactamase gene

AIM: To study the inhibitory effects of 10-23 deoxyribozyme (10-23 DRz) on Escherichia coli β-lactamase gene expression. METHODS: According to the gene sequence of Escherichia coli β-lactamase gene blashv-5, 10-23 DRz and antisense oligonucleotides (As-ODN) were designed and synthesized. 10-23 DRz, As-ODN or control oligonucleotides were respectively introduced into Escherichia coli by the method of electroporation. Following electroporation, bacterial viability in liquid medium contained ceftazidime was detected, bacterial β-lactamase expression was analysed by using IEF-PAGE and the β-lactamase band was measured with gel documentation-analyzing system. RESULTS: A600 in 10-23 DRz transfected Escherichia coli was lower compared with that in As-ODN transfected Escherichia coli (P<0.05). Bacterial β-lactamase expression in 10-23 DRz transfected Escherichia coli was also lower compared with that in As-ODN trnasfected Escherichia coli (P<0.01). CONCLUSION: 10-23 DRz specificly inhibits bacterial β-lactamase gene expression.     

Expression of hepatitis B virus core gene in Pichia pastoris

AIM: To study the expression of hepatitis B virus core gene in Pichia pastoris and to obtain high-level expressed recombinant HBcAg with good immunoreactivity and high specificity. METHODS: HBV core gene was amplified by PCR from plasmid pHBV1 which contained HBV whole DNA sequence. The PCR product was cloned into pGEM-T vector by TA cloning strategy. After confirmed by DNA sequence analysis, the gene of interest was inserted into the yeast expression vector pPIC9. The recombinant plasmid pPIC9-cAg was constructed and transformed into GS115 by electroporation. The recombinant yeast GS115 was induced by 0.5% methanol. The expressed product was analysed by SDS-PAGE,Western blot and ELISA. RESULTS: The restriction analysis and DNA sequence analysis proved that HBV core gene had already been cloned to yeast expression plasmid pPIC9. The expressed HBcAg existed in SDS-PAGE. Good immunoreactivity and high specificity of the recombinant HBcAg have been proved by ELISA and Western blot. The titre of the recombinant HBcAg in the cell lysate was 1∶12 800. CONCLUSION: The recombinant plasmid pPIC9-cAg was successfully constructed. The recombinant HBcAg with good immunoreactivity and high specificity was successfully expressed in Pichia pastoris expression system and can be applied to further developing HBcAb immunoassay.     

食用菌遗传转化研究进展

食用菌遗传转化的方法主要有PEG法、电激法、基因枪法、限制酶介导法，农杆菌介导法；采用的启动子有ras启动子和gpd启动子，采用的筛选标记有营养缺陷型标记，抗生素抗性筛选标记，除草剂和杀菌剂抗性筛选标记，代谢产物抗性筛选标记，本文综述了这些遗传转化方法以及启动子，筛选标记的最新研究进展。     

Transfection and expression of murine interleukin-12 gene in B16F10 melanoma cells

AIM:To study the expression of murine interleukin-12(mIL-12) gene in B16F10 melanoma cells.METHODS:mIL-12 gene was inserted into pcDNA3.1 eukaryotic expression vector by DNA recombination and then transfected into B16F10 melanoma cells by electroporation. The integration and expression of mIL-12 gene in B16F10 melanoma cells were detected by PCR, RT-PCR and Western blot after positive cell clones had been screened.RESULTS:mIL-12 gene was confirmed to have been transfected and expressed in B16F10 cells on three levels of DNA, mRNA and protein.CONCLUSION:mIL-12 gene can successfully be transfected and expressed in B16F10 melanoma cells in vitro, which lays a foundation for the further investigation of mIL-12 gene tumor vaccine.     

担子菌遗传转化研究进展

担子菌遗传转化目前主要采用电激法和PEG法。本文着重介绍自第一例担子菌的遗传转化开始,担子菌中转化系统的构建和转化的一般特点;并列出了截止到1996年9月报道的一些实例,说明了担子菌遗转传化的理论价值和应用价值。     

外源DNA片段导入草菇原生质体的研究

以平菇DNA为供体,草菇原生质体为受体,用PEG,CaCl_2作诱导剂,将平菇DNA导入草菇原生质体,选育出V_(157-1)菌株,该菌株在菇型、温型、酶活力及生物转化率上与亲本V_(157)有显著差异。本研究为食用菌育种提供了一条新的途径。     

植物转基因技术及其应用

本文综述了五种重要的植物遗传转化方法(农杆菌介导法,聚乙二醇法,基因枪法,花粉通道法和电激穿孔法)及其应用,并阐述了植物转基因技术在抗除草剂,抗逆性和品质改良等方面的研究进展。     

香菇ras和gpd启动子的克隆与功能鉴定

以香菇基因组DNA为模板,利用PCR技术克隆了香菇1个ras启动子Lras,2个gpd启动子Lgpd1和Lgpd2.这3个启动子的大小分别为715bp、1056bp和611bp,与已报道的ras和gpd启动子的同源性很强.对这3个启动子的分析结果表明,它们都含有丰富的启动子顺式作用元件.将这些启动子片段分别与GUS基因连接构建了表达载体,并将这些表达载体导入草菇菌丝体中,检测其活性,最终显示3个片段都有启动GUS基因表达的能力,其中Lgpd1启动子活性最强,Lras启动子次之,Lgpd2最弱.     

Role of Slit2/Robo1 signaling in development of neural tube and somites in early chick embryos

AIM: To investigate the effects of Slit2/Robo1 signaling on the development of neural tube and somites in early chick embryos. METHODS: Plasmid DNA was injected into the lumen of the neural tube from dorsal side of HH10 chick embryo using microinjection, and then in ovo electroporation was performed at half-side of neural tube while another side served as control. Subsequent 10-hour incubation was carried on after transfection until the development of neural tube and neural crest cells migrating to somites were investigated using the methods of immunofluorescence and in situ hybridization. RESULTS: Blocking Slit2/Robo1 signaling resulted in abnormal development of neural tube, while the expression of Slug and neural crest cells migrating to somites pathway were abnormal as well.CONCLUSION: Slit2/Robo1 signaling can affect the expression of Slug and play an important role in the fusion of neural fold, the trajectory of generation and migration of neural crest cells, and the differentiation of somites in early chick embryos.     

Transfection of WT1 gene isoforms and establishment of leukemia cell lines stably overexpressing WT1 gene

AIM: To transfer 4 full-length WT1 isoforms cDNA into the leukemia cell line NB4 so as to provide a cell model for studying the WT-1 gene function. METHODS: The eukaryotic expression recombinant vectors for WT1 isoforms (pCB6+/WT1) were introduced into the leukemia cell line NB4 by electroporation. The positive cell clones were screened by G418 culture. The integration of WT1 gene isoforms in NB4 cells as confirmed by PCR. The mRNA and protein of WT1 were detected by RT-PCR and Western blotting. RESULTS: WT1 gene isoforms were successfully transferred into NB4 cells. WT1 mRNA and protein expression in the G418-selected cells increased remarkably compared with the control. CONCLUSION: WT1 gene isoforms were effectively transferred into NB4 cells by electroporation and stably expressed in the transfected cells.