叶绿体遗传转化：植物导入外源基因的新途径

叶绿体转化系统,独立于传统的核转化,为植物导入外源基因提供了新途径。它有如下优点：超量表达目的基因;以定点整合方式导入外源基因从而消除了位置效应及基因沉默;具原核表达方式,能以多顺反子的形式表达多个基因;母系遗传方式可防止基因扩散;基因产物区域化并能提供适于某些产物发挥功能的小环境等。随着这项技术在越来越多的领域发挥作用,其优越性逐渐得到认同。本文着重对此技术的特点、发展及应用作一综述。     

高等植物的质体基因转化

质体基因转化技术由于其独特的优越性,已开始成为植物基因工程中新的研究热点。本文对质体基因转化技术所面临的几个关键性问题,包括外源基因导入方法、外源基因的整合及表达、质体转化体的有效筛选标记等进行了讨论,同时对质体基因转化的优越性及其应用与发展前景进行了综合评述。     

影响枯草杆菌原生质体转化的因素

原生质体转化是将外源基因导入细菌的主要方法之一,其转化频率受制于多种因素。本研究以激BacillussubtilisDB104为宿主菌,以枯草杆菌的高表达型质粒pNQ122为外源DNA,研究了原生质体再生率、原生质体浓度和用于制备原生质体的细胞生长期对转化频率的影响,获得了在该系统中实现高频率转化的条件。该转化条件使外源基因在多个B.subtilis菌株中的转化成为可能,并使从B.subtilis中筛选中性蛋白酶基因获得成功。     

高等植物叶绿体基因组的转化

介绍了高等植物叶绿体基因组转化技术的原理和优点,外源基因导入叶绿体基因组的方法,外源基因与叶绿体基因组的整合及其表达,常用的叶绿体基因组转化的筛选标记基因及其去除的研究进展.     

苏云金芽孢杆菌杀虫蛋白基因克隆及油菜叶绿体遗传转化研究

向细胞核导入外源基因的核转化技术是植物基因工程的主要方法。然而,外源基因表达效率低,表达不稳定,基因易失活和因随机插入而造成的位置效应等是该方法不足之处。而且,由于外源基因可随花粉扩散,细胞核转化体的生物安全性问题已在全球范围内引起世人的关注和担忧。将外源基因导入叶绿体基因组有望克服细胞核转化中存在的某些弊端。油菜作为世界上重要的油料作物,其叶绿体遗传转化研究还未见报道。本研究从苏云金芽孢杆菌克隆得到野生型杀虫晶体蛋白基因,构建了用于油菜叶绿体定点转化的植物表达载体,并用基因枪法将杀虫蛋白基因导入油菜,…     

影响苹果离体叶片早期转化效率因素的研究

利用gus为报告基因,通过统计gus基因瞬时表达的GUS 位点数和形成的愈伤组织,快速检测外源基因是否导入植物细胞及转化效率,以及对苹果转化早期阶段的影响因素。结果表明,A281对苹果叶片的浸染率比LBA4404浸染率要高,转化效率要好;叶片近轴面接触培养基转化效率较好,显著高于远轴面;整叶划痕为最佳切割方式,有利于提高苹果叶片转化率;苹果基因型、菌液浓度和共培养天数对不同基因型苹果的转化效率影响很大。     

低能氮离子注入大肠杆菌诱发的生物学效应研究

研究离子注入的诱变和导入外源DNA的生物学效应,用低能氮离子注入处理大肠杆菌野生型菌株MC4100A,将含水母绿色荧光蛋白基因的质粒导入细胞中。实验结果表明,在一些转化子中绿色荧光蛋白的表达或折叠受到了影响,一些转化子丧失了分裂后分离能力,且细胞膜有一定程度的损伤,为进一步研究微生物细胞分裂和蛋白质折叠机理提供了菌株。故低能氮离子注入对微生物细胞的诱变效应和导入外源DNA效应的有机结合将使离子注入技术在生物学基础研究中有着广泛的应用前景。     

转双价抗虫基因大白菜新种质的获得及其抗虫性分析

采用超声波辅助花粉介导方法,将双价抗虫基因BmkIT-Chitinase导入早熟型大白菜自交系20-19-3,最终获得了5个转基因大白菜优良自交系纯合株系Z1-5、Z2-7、Z9-6、Z11-6和Z20-13;以转基因大白菜株系和非转基因对照植株为材料,对BmkIT-Chitinase基因在大白菜中的遗传规律、基因表达及抗虫性进行进一步分析。结果显示:(1)转化株后代多代(T_1~T_4)PCR、Southern blotting等分子跟踪检测表明,目的基因已成功导入受体植株,且能够稳定遗传;用该转基因方法对大白菜进行基因转化所获得的转基因植株分析显示,外源基因多数以多拷贝形式整合于核基因组,少部分外源基因以单拷贝形式整合。(2)Elisa分析结果证明,所导入的外源基因可高效表达,T4代株系新鲜叶片中表达产物量最高达到0.069μg·g~(-1)左右。(3)转基因株系田间抗虫性统计分析表明,转化株系与对照在抗虫性方面有显著差异,其对小菜蛾及菜青虫抗性普遍提高2~3级。研究认为,转BmkIT-Chitinase基因大白菜中BmkIT-Chitinase基因的表达可有效提高大白菜的抗虫性。     

高等植物的质体基因转化

质体基因转化技术由于其独特的优越性,已开始成为植物基因工程中新的研究热点。本文对质体基因转化技术所面临的几个关键性问题,包括外源基因导入方法、外源基因的整合及表达、质体转化体的有效筛选标记等进行了讨论,同时对质体基因转化的优越性及其应用与发展前景进行了综合评述。     

远缘杂交在转移有益基因创造小麦新种质中的潜力

本文概述了小麦远缘杂交技术的发展以及这些技术的应用对以染色体易位方式转移有益基因到普通小麦中的影响。通过对小麦远缘杂交技术的总结得出,普通小麦由于本身的多倍性,对导入的外源基因具有较强的调节能力,是适宜外源有益基因导入的良好受体。而以染色体易位方式转移有益基因是创造小麦新种质的有效方法之一,许多研究也表明以染色体易位导入的外源有益基因更利于表达。近几年,随着细胞遗传学以及其它生物技术的发展,对小麦族进化途径和染色体间的亲缘关系进一步明确,从而更便于进行易位导入的技术选择,也使得染色体易位鉴定方法更趋完善。现在已有更良好的外源导入的工具和方法,使多基因控制的外源优良性状导入成为可能。在小麦远缘杂交中染色体易位所具有的上述优势,在育种实践中逐步显示出来,为开拓小麦种质资源开创了一条新的途径。     

外源基因在莱茵衣藻叶绿体中的表达

把莱茵衣藻(Chlamydomonas reinhardtii)叶绿体作为生物反应器来表达外源基因具有广阔的应用前景。人们利用莱茵衣藻叶绿体表达体系已成功表达多种重组蛋白,其中包括人类药用蛋白。综述了莱茵衣藻叶绿体转化的方法、影响外源基因表达的主要因素以及外源基因在莱茵衣藻叶绿体表达研究进展。     

Development of DNA-mediated transformation systems for plants

The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed. Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants. Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation. A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates. Developing a more ubiquitous transformation system for monocotyledons is further from fruition. Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.     

水稻遗传转化研究进展

水稻遗传转化的主要方法,包括农杆菌介导法,基因枪法,电激法,PEG法等,论述了用于水稻转化的各种外植体,影响水稻遗传转化和植株分化的各种因素,各种标记基因及相应的选择方法和外源基因在转基因水稻中的遗传规律,比较了常用启动子在水稻中的表达强度,一些已转移到优良水稻品种中的有益农艺性状基因,如抗病、抗虫、抗除草剂等,介绍了它们在水稻品种改良中所发挥的作用。     

根癌农杆菌介导的水稻转化研究进展

农杆菌介导的水稻基因转化是水稻基因转化的热门。本文对由农杆菌介导转化获得的水稻品系（品种）,影响农杆菌介导转化的因素,农村菌浸染的方法,外源基因的检测和遗传等方面作综合论述,并提出了农杆菌介导转化水稻的前景。     

利用PEG法建立药用真菌灵芝的转化系统

本文首次建立了一种通过PEG转化缓冲液将外源基因转入灵芝的方法,转化频率约为5~6个/mg(抗性转化子/质粒+107个原生质体)。转化子在不含HmB的培养基上经5代以上的继代培养后仍可以稳定表达HmB抗性。Southernblot检测证明外源基因已经整合到了灵芝的基因组DNA中。本研究为通过基因工程手段定向、快速改良灵芝药用品质以及利用灵芝发酵方法生产一些具有重大经济价值的外源蛋白等应用奠定基础,并且也有助于我们进一步了解灵芝这一大型真菌中的基因的表达调控机制。     

Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration

A white-rot basidiomycete Ganoderma lucidum has long been used as a medicinal mushroom in Asia, and it has an array of enzymes important for wood degrading activity. There have been many reports about the ingredients which show health aiding effects. In order to analyze gene functions and introduce foreign genes into this fungus, genetic transformation is required. We have successfully transformed G. lucidum to geneticin resistance using pJS205-1 which has the antibiotic resistance genes against geneticin and phosphinothricin. Many different mutants have been generated during the transformation by restriction enzyme mediated integration, and the transformation yield was 4-17 transformants (microg plasmid DNA)(-1). The plasmid was integrated stably into the recipient chromosome, which was confirmed by PCR with the plasmid-specific primers.     

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.     

叶绿体转化体系研究进展

叶绿体转化具有表达效率高,安全性高,遗传稳定,以及多基因可以同时转化等特点。真核或原核的外源基因都能在叶绿体中进行成功表达,至少有20种植物成功的进行了叶绿体转化。叶绿体作为生物反应器具有广阔的应用前景。     

Binary vectors for efficient transformation of rice

We constructed binary vectors that were designed for transfer and expression of a gene into rice chromosomes. The binary vectors contained the hygromycin-resistance gene for selection of transformants and multiple-cloning sites within the transfer DNA. In addition, vectors were designed to express foreign genes using four kinds of promoters. We also report a procedure for efficient transformation of rice plants using scutellum-derived calli and theAgrobacterium strain LBA4404.