家畜繁殖生物技术研究现状及趋势

牛胚胎移植技术已趋于成熟。我国新疆牧科院用一步细管法移植牛冻胚受胎率达41%;牛和羊鲜胚四分胚移植也产下1头犊牛和6只羔羊。家畜体外受精,因卵子体外成熟和受精卵体外培养尚不过关,目前仍停留在实验室阶段。胚胎性别鉴定,1990年Koopman发现单拷贝基因,该基因是Y染色体的性决定区,命名为SRY,可利用PCR技术制成雄性特异DNA探针盒,进行马、牛、羊、猪早期胚胎性别鉴定。北京农学院等单位用PCR扩增牛SRY序列进行奶牛胚胎性别鉴定准确率达100%。英、日、法等国已获得牛胚胎细胞核移植后代;我国也获得1只核移植兔。北农大和新疆牧科院合作以绵羊精子为载体导入牛生长激素基因rMTbGH DNA成功,外源基因整合率为3%。     

肉兔双列杂交遗传效应的分析

本试验采用了５品种完全双列杂交，用Ｈａｖｅｙ程序对德国花巨兔（Ｇ）、比利时兔（Ｂ）、新西兰白兔（Ｎ）、加利福尼亚兔（Ｃ）、丹麦白兔（Ｄ）的主要生产性能进行了遗传效应分析。首次采用加性——显性模型分析了优势杂交组合优势性状的基因效应值与杂种优势率的关系，对杂种优势机理作了一定探讨。结果表明：最佳杂交父本、母本分别为Ｇ（♂）或Ｎ（♂）、Ｎ（♀）；最优杂交组合为ＧＢ、ＮＧ。此外，ＧＮ、ＤＮ、ＧＣ、ＢＣ也不失为优秀组合     

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Effects of blocking the expression of PKARⅠβ gene with antisense nucleotide on Shuang long-Jie gu Pill (SLJGP) containing serum osteoblast proliferation

AIM: To further investigate the role of PKARⅠβ in the growth-promoting effects of shuang long Jiegu pill (SLJGP), a Chinese medicine, on cultured osteoblasts. METHODS: pcDNA- antiPKARⅠβ, a recombinant expressing the antisense sequence of PKARⅠβ, was constructed and transformed HFOB1.19 by lipofectin. MTT was undertaken to assess the cell growth with the treatment of high dosage of SLJGP containing serum. RESULTS: Antisense gene blocked the growth-promoting effects of SLJGP containing serum on HFOB1.19. CONCLUSION: The function of SLJGP is closely related to cAMP-dependent protein kinase A.     

山羊痘病毒SS株5个ANK基因缺失的重组病毒构建

为研究锚定蛋白基因(Ankyrin,ANK)对山羊痘病毒的影响,试验采用融合PCR和Overlap PCR技术扩增山羊痘病毒SS株5个ANK基因(ANK010、ANK138、ANK 140、ANK141.2和ANK145)两端侧翼序列和绿色荧光蛋白(GFP)基因,并将其产物连接Trans1-T 1载体构建ANK缺失的转...     

桑树花青素合成酶(ANS)基因的克隆及在2种果色桑树中的表达特征

花青素合成酶(anthocyanidin synthase,ANS)是植物花青素生物合成途径末端的关键酶,催化无色花色素到有色花色素的转变。从广东桑品种粤椹大10中克隆得到一条ANS序列,其ORF为1 077 bp,编码358个氨基酸,预测蛋白质分子质量为41 kD,等电点为5.62,具有2-酮戊二酸双加氧酶的保守结构域。多重序列比对表明物种间的ANS序列高度保守,系统进化树中桑树ANS与芍药科、芸香科、葡萄科等植物的同源序列聚在一个类群上。RT-PCR检测桑树ANS在结紫色果的粤椹大10的幼叶和桑椹中特异性表达,并且随着果色加深其表达水平呈上升趋势,而在结白色果的桑品种珍珠白的幼叶和桑椹中检测不到ANS的表达,暗示桑树ANS在桑椹的颜色形成中起到重要作用。     

小菊'类锌指蛋白'基因植物表达载体构建及对烟草的转化

为了建立耐盐碱小菊(Chrysanthemum morifolium)中克隆到的'类锌指蛋白基因'CmSTZF(DQ864730)的烟草遗传转化体系,并研究其抗逆特性。本研究借助Gateway技术构建了小菊'类锌指蛋白'基因CmSTZF的过量表达载体pH7WG2D-CmSTZF,并以烟草NC89(N.plumbaginifolia'NC89')的叶片为试材,通过农杆菌介导的转化,成功地建立了烟草遗传转化体系,经PCR-Southern鉴定获得CmSTZF的转基因烟草株系。     

Alteration of apoptotic susceptibility and bcl-2 gene in malignant transformation of human bronchial epithelial cells

AIM: To demonstrate the susceptibility of cell apoptosis varies during the progress of cell malignant transformation from human being in vitro. METHODS: A SV₄₀T-transfected human bronchial epithelial immortalized cell line (called M) was selected in this work, which has acquired some characteristics of malignant transformation at the later passage. The alterations of apoptosis and bcl-2, P53 genes between early and later passage of M cells were investigated by means of TDT labeling in situ, chromosome FISH, RNA and protein testing, etc. RESULTS: Incidence of apoptosis induced by cis-platin was significantly lower in later than in early passages of M. Levels of bcl-2 mRNA and protein in later passages were higher than early passages of M, and overexpression of bcl-2 was accumulated following the development of cellular malignancy. P53 protein level was as high in early as in later passages. CONCLUSION: Overexpression of bcl-2 decreases the cellular sensitivity to apoptotic inductors plays an important role during progress of carcinogenesis in human bronchial epithelial cancers. The inactivation of P53 protein in the SV₄₀-T transfected M cell line may be one of reasons of bcl-2 overexpression, but not associated with the accumulation of bcl-2 expressed level during cell transformation.     

Expression of phenylalanine ammonia-lyase cDNA from rice in E. coli BL21DE3

AIM: To study the expression and its kinetics of rice phenylalanine ammonia-lyase gene encoding into E. coli as the basis of treatment for phenylketouria. METHODS: The phenylalanine ammonia-lyase-1-cDNA(rPAL-1-cDNA) from rice was recombined into E. coli high expression vector pET-28c and transformed into E. coli host strain BL21DE3. Engineering bacteria was then inducted by isopropyl-β-D-thiogalactoside (IPTG) for 1, 3, 5, 7 hours, in order to obtain high level expression. RESULTS: After induction, the expression level of fusion protein was 21.40%, 30.60%, 35.40%, 35.43% respectively. The fusion protein exhibited a band of 78.6 kD on SDS-PAGE analysis, but was not found in controls.The target protein was mainly existed in the form of inclusion body. CONCLUSION:Rice PAL gene expressing E. coli was established by gentic engineering technique.