用激光微束法将外源基因导人水稻细胞的研究

将外源基因导入植物细胞是植物基因工程的关键步骤之一。用激光微束技术转化动物细胞已经取得了成功,并证明外源基因已整合到细胞染色体上。最近又证明激光微束可穿透植物细胞壁和细胞膜,将pBR322DNA导入植物细胞和细胞器。本实验在利用激光微束技术将外源基因导入水稻培养细胞上进行了探索。 所用外源DNA是pBI121质粒,该质粒带有CaMV35启动子和β-葡萄糖苷酸酶(GUS)基因,用     

激光微束诱导pSV—β质粒导入Hela细胞的研究

本文报道了用一套Ｎｄ：ＹＡＧ紫外激光微束系统研究基因转移。我们以ｐＳＶ－β质粒（β－半乳糖甙酶基因）作为报道基因,利用外激光微束将其导入Ｈｅｌａ细胞中,通过组织化学染色检测到质粒在Ｈｅｌａ细胞中获得表达,在最佳的激光辐照条件下,Ｈｅｌａ细胞的导入表达成功率在８０％以上。本工作是国内首次报道激光微束诱导外源基因进入动物细胞中获得表达,为激光微束技术在动、植物细胞高效率导入外源ＤＮＡ的研究中摸索到一套     

一种新型的激光微束细胞打孔仪系统

研究成一种高性能的激光微束细胞打孔仪。YAG腔外倍频激光经空间滤波器、聚焦物镜作用于细胞,产生小于1μ的打孔直径,一个高倍率(约2000~X)电视监测系统作为细胞打孔及观测用;设计了一种长工作距(c′≥15mm),短焦距(f_o=6.25mm)的特殊聚焦物镜。     

利用微束激光穿刺法将抗真菌基因导入棉花的研究初报

以棉花感受态萌动种胚作为外源基因转化的受体,用激光微束穿刺法将β-1,3-葡聚糖酶及几丁质酶双价基因导入棉花,所构建的植物表达载体pB IBGC携带有筛选标记npt-Ⅱ(新霉素磷酸转移酶)基因。激光转化处理的种胚经卡那霉素筛选,已经获得抗性小植株。研究了微束激光转化法用于棉花感受态萌动胚转化预培养的时间、高渗液对材料的处理等。研究表明:用微束激光转化处理种胚是一种操作简便、重复性好的转化方法,用该法可将外源基因导入植物感受态萌动胚,避开植株离体再生的困难。     

激光微束诱导pSV－β质粒导入Hela细胞的研究

本文报道了用一套Ｎｄ：ＹＡＧ紫外激光微束系统研究基因转移。我们以ｐＳＶ－β质粒（β─半乳糖苷酶基因）作为报道基因,利用紫外激光微束将其导入Ｈｅｌａ细胞中,通过组织化学染色检测到质粒在Ｈｅｌａ细胞中获得表达,在最佳的激光辎照条件下,Ｈｅｌａ细胞的导入表达成功率在８０％以上。本工作是国内首次报道激光微束诱导外源基因进入动物细胞中并获得表达,为激光微束技术在动、植物细胞高效率导入外源ＤＮＡ的研究中摸索到一套经验,也将有力地促进这一技术在细胞工程方面的应用。     

发刊词

当代,科学技术迅猛发展,呈现出既高度分化又高度综合的特点。一大批新兴学科相继涌现,激光生物学正是在这个大潮中形成和发展起来的边缘学科。它是激光技术向生物学渗透,是激光医学、激光育种等应用学科的基础。它一经诞生,就得到了广泛的应用。目前,国内外利用激光研究生命物质运动,已进入细胞水平和分子水平,用激光微束技术实现了切割染色体、核仁组织者的定位、遗传缺失和动物卵细胞融合,诱发微核仁形成;用激光微束技术将外源基因导入小麦和水稻细胞以及向蚕卵细胞注入外源基因等方面都取得了进展。可以预见在生物工程技术领域中将会逐渐形成一个新的分支——激光生物工程,它的作用将与酶工程媲美。     

激光微束诱导鲶鱼受精卵融合的研究

本文论及激光微束诱导胡子鲶（C．fuscus）受精卵融合的一种技术及其对鲶鱼遗传变异和生长发育的影响。实验表明：一些融合卵发育非常健全；实验所得成年鱼中，有一条体色变为白色、一条变成略带淡紫色，二条O形和O须都有变化；融合鱼体长比对照（未经激光处理）鱼约长1．5倍，体重比对照鱼约重2倍；抗寒性能比对照亦提高1．5℃～2℃．     

微束激光转基因技术研究进展

本文叙述了激光微速穿刺法导入外源ＤＮＡ的基本原理及其有关影响因素等。利用该转化方法,现已成功地获得了多种动植物细胞外源基因的转化。实验证明,激光微束穿刺转化技术简便有效、重复性好、靶体选择性强,对靶体无损伤等优点。随着转化技术自动化水平的提高。光镊技术的渗透和结合,激光微束转化技术将会得到更广泛的应用     

激光微束在植物外源基因转化上的应用

本文概述了近年来激光微束在植物外源基因转化上的最新进展,激光微束转化法的关键技术及应用。并对其今后的应用前景作了展望。     

将菜豆几丁酶基因导入苹果砧木的研究

以苹果砧木八楞海棠（Ｍａｌｕｓｍｉｃｒｏｍａｌｕｓ）为实验材料,用微束激光穿刺法将菜豆几丁酶基因导入苹果叶片,成功地获得了转基因植株。实验结果表明,以苗龄２６天的小苗上部刚展开的叶片再生频率可达１００％。经高渗缓冲液预处理后用激光微束穿刺导入外源ＤＮＡ可得到较高的转化频率。在１５０个再生叶片中经卡那霉素筛选得到２５株绿苗,经ＰＣＲ扩增检测,３株呈阳性结果,表明外源几丁酶基因已整合到苹果的基因组ＤＮＡ中。     

Artificial insemination of the American lobster (Homarus)

A technique has been developed for artificially inseminating freshly molted female lobsters (Homarus). Using the criterion that eggs were fertilized if they showed development of normal cleavage patterns, at least 49.5% of the inseminated females extruded fertilized eggs. Sixty-two percent of the fertilized eggs developed to the eyespot stage, and 26.6% of these subsequently hatched. Eggs were not fertilized in 7.7% of the extrusions. In 42% of the samples, it was not possible to establish whether fertilization had occurred because either samples were collected prior to initiation of cleavage or eggs were too poorly preserved to assess cleavage with confidence. It is probable that many of the eggs that were evaluated as equivocal were also fertilized. This technique should be valuable in the experimental and commercial aquaculture of lobsters and might be directly applicable to other crustaceans.     

激光微束切割小冰麦异附加系染色体的研究

利用激光微束,并选用适当的功率密度可将小冰麦异附加系花粉细胞染色体切割成2～3个片段,这个技术的建立为激光微束应用于染色体片段DNA微克隆及基因定位提供可能。     

Oocyte Maturation and Furrow Formation in an Unfertilized Egg by Fusion with a Fertilized Egg or Blastomeres in the Ascidian, Ascidia sydneiensis divisa

A technique for fusing an ascidian egg with blastomeres using a chemical fusiogen was established and then used to identify cytoplasmic factors that regulate the process of oocyte maturation in ascidian eggs. Unfertilized eggs fused with fertilized eggs or blastomeres in hypotonic artificial sea water containing 20% polyvinyl alcohol within 10 min. After fusion polar bodies were extruded from the unfertilized portion of the fused eggs. Furrows were formed not only in the fertilized portion but also in the unfertilized portion in the fused eggs. No polar body extrusion and furrow formation occur in either portion of fused unfertilized eggs. These results suggest that fertilized eggs and blastomeres contain a factor that induces oocyte maturation. Polar body extrusion and furrow formation were not suppressed in the fertilized portion of fused eggs, suggesting that unfertilized eggs do not contain a factor that inhibits oocyte maturation.     

SPERM MIXING IN THE COCKROACH DIPLOPTERA PUNCTATA

Females of the viviparous cockroach Diploptera punctata store sperm from their first mating, and do not remate until after giving birth to their first batch of young. The irradiated male technique was used to determine the outcome of sperm competition in the second batch of eggs of females mated sequentially to normal and irradiated males. It is estimated that the second male to mate with a female fertilizes approximately two thirds of the eggs in a female's second batch of eggs. Direct evidence for sperm mixing was obtained. Undeveloped eggs (fertilized by irradiated sperm) and developing embryos (fertilized by normal sperm) were found interspersed throughout oothecae that were extruded from females, demonstrating that normal and irradiated sperm were released from the spermathecae at oviposition and that they competed for fertilizations.     

细胞松弛素B对鲍受精卵超微结构的影响

细胞松弛素B（CB）作为一种微丝解聚剂,可用于诱导贝类三倍体,但由于其毒副作用导致了受精卵死亡和发育异常。通过透射电镜观察比较了皱纹盘鲍（Haliotis discus hannai)正常受精卵与CB处理后的受精卵亚显微结构的变化,结果显示,CB对线粒体等细胞器的破坏,造成了受精卵的代谢缺陷,是导致鲍受精卵孵化率低的原因之一。     

Assisted fertilization by zona drilling: a mouse model for correction of oligospermia

A micromanipulation apparatus was used to produce holes in the zonae pellucidae of unfertilized mouse oocytes. A microneedle loaded with acid Tyrode's solution was brought into contact with the zona surface, and positive flow was used in conjunction with mechanical pressure to cause a localized dissolution of the zona. Treated eggs were then fertilized in vitro in comparison with control cells. The zona drilling procedure decreased the sperm count required to achieve fertilization by a factor of approximately 100. The rate of polyspermy in zona-drilled oocytes was not greater than in controls, and oocytes fertilized after drilling, when implanted into pseudopregnant foster females, developed to term at the same rate as controls. The results demonstrate that zona drilling is a safe, effective method of increasing the efficiency of fertilization in vitro and may be useful both in agriculture and medicine for conferring fertility upon males with low sperm counts.     

On the synthesis of glutamine before and after fertilization of the sea urchin, Strongylocentrotus purpuratus

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, have a much lower capacity for glutamine synthesis than do fertilized eggs. This difference is not caused by an alteration of glutamine synthetase activity attendant upon fertilization. Neither the specific activity of glutamine synthetase nor its pattern of activation by divalent metal ions is affected by fertilization. The enzyme from both fertilized and unfertilized eggs is activated by α-ketoglutarate and inhibited by ultimate end products of glutamine metabolism. This type of regulation is similar to that seen with many other eucaryotic glutamine synthetases.Unfertilized eggs take up less glutamic acid than do fertilized eggs when the amino acid is presented at high concentrations (12.5 mM), whereas there is no difference in glutamic acid uptake at low concentrations (5 μM). Under conditions where glutamate uptake is identical, unfertilized eggs are dependent upon exogenous ammonia for glutamine synthesis in vivo; fertilized eggs are able to synthesize glutamine in the absence of added ammonia. Thus, our data suggest that the increased capacity for glutamine synthesis after fertilization is related to an increased availability of the substrate, ammonia.     

On protein fractions and inorganic ions in sea urchin eggs,unfertilized and fertilized

Frozen and dried unfertilized or fertilized eggs were subjected to extraction by 1-N KCl solution according to the method inaugurated by Mirsky.In confirmation of Mirsky's results a certain protein fraction seemed to become insoluble in normal fertilization of the egg.The differences proved almost to vanish if the eggs prior to fertilization were subjected to trypsin treatment which removes the vitelline membrane and thus inhibits the formation of a continuous fertilization membrane.An obvious correlation was found between the percentage of extracted proteins and the salt content of the egg samples (fig. 1). This is particularly high in frozen and dried fertilized eggs with the fertilization membrane normally formed. This probably accounts entirely for the previously reported differences between unfertilized and fertilized eggs.Extraction of the frozen and thawed eggs with distilled water gave the same general picture as extraction with N KCl. Here also the solubility of the proteins is correlated with the salt content of the samples.The dialyzed water extracts gave a strong precipitation upon addition of Ca (optimum at 0.0125-0.025 M). Freezing and thawing of the extracts likewise cause a precipitation of a certain fraction.The fraction which becomes insoluble in the presence of high salt content of the sample and that precipitated from extracts by additions of Ca or by freezing and thawing seem to be identical. It contains the soluble nucleoprotein of the eggs.No changes in Ca or Mg content was found to occur upon fertilization of the egg.     

激光照射对中华大蟾蜍早期胚胎染色体的影响

应用YAG倍频激光器对中华大蟾蜍四细胞期胚胎进行照射,观察胚胎的生长、发育,并对激光照射致畸的胚胎在尾芽期用压片法进行染色体分析。实验结果表明：激光照射后正常的胚胎其染色体无变化,激光照射致畸的胚胎其染色体有丢失和加倍等变化。