拟衣藻系统分类新证据

拟衣藻 (Chloromonas)与衣藻属 (Chlamydomonas)的亲缘关系及分类学位置在藻类学界一直没有定论。其重要原因是单细胞鞭毛类是否具有蛋白核这一特征在系统分类学上具有重要意义 ,而光镜形态与色素体上具 1到多个蛋白核的衣藻极其相似的拟衣藻 ,其不具蛋白核这一特征的稳定性受到许多学者的怀疑。本文观察并报道了中华拟衣藻 (ChloromonasSinica)的超微结构 ,通过对拟衣藻与衣藻超微结构的探究和比对 ,发现除了从孢子时期开始的整个生活史中 ,中华拟衣藻都不具蛋白核外 ,无论显微还是超微结构 ,拟衣藻与衣藻都显示出高度的相似性。从而提出在尚未获得更多资料之前 ,将拟衣藻从衣藻属分离出来成为独立的属较为合适     

拟衣藻属一新种——中华拟衣藻及其生活史的研究

报道了团藻目衣藻科拟衣藻属一新种。此种从中国科学院武汉植物研究所一浇园粪缸中采得,在分离室内单种培养过程中,对其形态学和生活史进行了研究。营养细胞具有多个盘状色素体,与拟衣藻属已知的种类有明显的差别。经培养观察,作者发现除细胞分裂、无性生殖外,还有同宗异配式有性生殖,在生活史各阶段该藻中绿体中均未见蛋白核,因此,拟衣藻作为分类单元是一个有效的属。     

衣藻叶绿体表达系统的研究

真核藻类作为一种新型的蛋白表达系统,因其培养方法简单,成本低廉并且能大规模繁殖,最近几年成为人们关注的焦点.作为一种模式生物,单细胞的真核生物莱茵衣藻已经成为人们研究的重点.外源蛋白不仅能在衣藻核中进行表达而且也能在叶绿体中表达,但衣藻叶绿体的表达系统较之核表达有巨大的优越性.在到目前为止,已经有许多的药用蛋白在莱茵衣藻的叶绿体中成功表达的报道,证明了莱茵衣藻叶绿体作为生物反应器的能力.将对衣藻叶绿体的表达做详细的描述.     

莱茵衣藻细胞核转化体系研究进展

莱茵衣藻(Chlamydomonas reinhardtii)是一种三套基因组都可以进行遗传转化的模式生物,具有培养条件简单、生长速度快、光合效率高等优点,细胞核转化体系相对更为成熟,将其开发为生物反应器具有广阔的应用前景。该文对衣藻核基因组特点及转化机理、转基因衣藻的筛选方法、外源基因的表达以及影响因素等方面进行了综述。     

衣藻的细微结构

衣藻属于绿藻门、团藻目。它是绿藻中细胞结构特征的一个典型代表。大、中学植物教材中都以衣藻作为绿藻门的重要代表植物。本文根据目前电镜资料,描述如下。衣藻的细胞器衣藻虽然是较原始的单细胞真核藻类,但是它已具备了真核植物的各种细胞器。在光学显微镜下可以清楚地看到,在细胞壁内有包埋在细胞质中的大型杯状叶绿体和叶绿体腔内的细胞质及细胞核。在电镜下观察,细胞器与高等植物的相同。核膜具有双层膜结构、有核膜孔。细胞质中的内质网与核膜     

适用于本科实验教学的衣藻核遗传转化方法研究

对莱因衣藻衣藻核遗传转化关键技术中的衣藻对数生长期确定、玻璃珠性质及振荡操作环节进行了研究,建立了既简便、廉价,又稳定、有效的衣藻核遗传转化系统方法。该方法适用于高等院校本科生遗传学实验教学,其涉及的实验技术可成为培养学生综合实验技能的理想教学内容。     

基因编辑技术在莱茵衣藻中的应用进展

基因编辑技术已经成为功能基因组学和作物分子育种精准且有力的工具。莱茵衣藻(Chlamydomonas reinhardtii,简称衣藻)是光合作用、黑暗异养代谢、厌氧代谢和生物制氢、营养和能量代谢等研究领域的重要模式生物。近20年来,基因编辑技术,如锌指核酸酶(ZFNs)、转录激活物样效应物核酸酶(TALENs)、CRISPR/Cas9和CRISPR/Cpf1等的发展,更加推动了以衣藻为模式生物的研究。现对衣藻中的核基因组靶向基因编辑技术的应用及最新研究进展等进行总结,以期为衣藻相关领域的研究提供参考。     

衣藻(Chlamydomonas sp)中间纤维及核纤层存在的证实(简报)

衣藻(Chlamydomonas sp)是属于绿藻门的最低等单细胞植物,为典型的真核生物。迄今以衣藻为材料所作的有关细胞骨架方面的研究多集中在微管蛋白(tubulin)。C.J.Miller等曾以衣藻(Chlamydomonas reinhardtii)全蛋白与几种中间纤维抗体进行免疫印迹实验有阳性反应,但是衣藻中是否存在中间纤维与核纤层是不清楚的问题。衣藻中间纤维与核纤层的形态研究更未见报道。目前认为中间纤维-核纤     

衣藻和水绵

衣藻和水绵不仅因为是一种最常见的绿藻,同时也由于它们具有实际的经济意义,而为中学和大学教材所采用。衣藻常为鱼苗的食饵,同时,从生产的观点上看来,衣藻和水绵也具有经济意义。衣藻可做为某些鱼苗的食饵,水绵又常因其丝状体的大量繁殖而困陷鱼苗并致其窒息,是为一种鱼害。     

同源重组置换衣藻叶绿体chlL基因及其同质化鉴定

设计以 aad A基因置换衣藻 chl L基因并作为转基因衣藻筛选标记 ,将 chl L基因两侧的基因片段 clp P- trn L- pet B- chl L5′- 3′作为同源片段 ,构建衣藻叶绿体同源重组质粒 p64D.通过改进的基因枪法转化程序 ,将此质粒轰击入衣藻叶绿体 .经抗性筛选、暗培养及 PCR、Southern- blotting等鉴定 ,证实衣藻叶绿体基因组中大部分 chl L结构基因已被外源基因 aad A所置换     

Direct creation of marker-free tobacco plants from agroinfiltrated leaf discs

Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for β-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.     

Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes

The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.1 micromolar) of 1-naphthalene-acetic acid, pedicel strips from phenotypically aberrant plants regenerated two to three times more flower buds than explants from untransformed tobacco. Intermediate bud numbers were observed in transformants with a less extreme phenotype. The results can be explained by an increased sensitivity of the transformed explants to auxin with respect to flower bud regeneration. The effect of transformation on the auxin response is fully accounted for by the absence of a negative interaction of endogenous ethylene with 1-naphthaleneacetic acid, a phenomenon normally encountered in untransformed tissues. Three observations led to this conclusion. Application of 1 micromolar AgNO₃ to untransformed explants increased the number of flower buds to the level observed in transformed tissues but had no effect on transformed pedicel strips; exposure to 10 microliters per liter ethylene strongly reduced the response to auxin at all concentrations in untransformed explants but was almost ineffective in the transformed tissues; and endogenous ethylene synthesis occurred at the same rate in both types of explants.     

Morphogenesis and Auxin Sensitivity of Transgenic Tobacco with Different Complements of Ri T-DNA

Leaf explants of hairy root tobacco (Nicotiana tabacum) regenerants characteristically differentiate roots from the wound margins on hormonefree medium. The same response can be elicited on normal tobacco by culturing the explants in the presence of auxin. We show here that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones. Rather, an increased sensitivity to auxin is conferred to transformed cells by the left T-DNA (TL-DNA). Analysis of the morphogenetic behavior of transgenic tobacco plants obtained by transferring segments of TL-DNA cloned in a binary vector system allowed us to pinpoint TL-DNA genes responsible for this increased auxin sensitivity of hairy root tissues. Three genes (open reading frames 10, 11, 12) are responsible for the spontaneous rooting of leaf explants and confer to transgenic plants an exaggerated response to auxin.     

甜味蛋白thaumatin基因转入烟草的研究

利用基因工程技术 ,将分别克隆在两个不同载体上的甜味蛋白 thaum atin c DNA基因片段连接成一个完整的 c DNA基因 ,并将该基因克隆进 p BI12 1,构建成表达载体 p BI12 1- tha.通过冻融法导入农杆菌 ,农杆菌介导叶盘法转入烟草 ,经过组培 ,得到转基因的植株 .提取转基因烟草总 DNA,经 PCR,PCR- Southern和 Southern杂交证实 ,甜味蛋白基因已整合到烟草基因组中 .RT- PCR结果证明 ,thaumatin基因已在转基因烟草中转录成 m RNA,但SDS- PAGE和甜味尝试都表明 thaumatin基因在转基因烟草中没有表达出甜味蛋白     

人干扰素α-2b基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b(Human interferon α-2b,HuIFN α-2b)编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。     

Production of Fibrinolytic Enzyme in Plastid-Transformed Tobacco Plants

The earthworm fibrinolytic enzyme, which belongs to a group of serine proteases with strong fibrinolytic activity, has been used as an oral drug for prevention and treatment of thrombosis in East Asia. Fibrizyme is a fibrinolytic enzyme isolated from the earthworm Eisenia andrei. Here we report genetic engineering of tobacco plastids with stable integration of the fibrizyme gene into the tobacco chloroplast genome. A plastid transformation vector was constructed by introducing various regulatory elements into fibrizyme cDNA. This vector was delivered by particle bombardment into tobacco leaf explants and plastid-transformed plants were subsequently regenerated into whole plants through several rounds of selection. We confirmed stable integration of the fibrizyme gene into the tobacco plastid genome by PCR and Southern blot analyses. Northern and Western blot analyses revealed that mRNA and protein of recombinant fibrizyme were highly expressed in transformed tobacco plants.     

An exogenous chloroplast genome for complex sequence manipulation in algae

We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.     

Development of broad-spectrum insect-resistant tobacco by expression of synthetic cry1Ac and cry2Ab genes

Efficacy of two newly synthesized cry1Ac and cry2Ab genes was checked in tobacco before their expression in cotton. Both genes were artificially synthesized and codon optimized with respect to cotton-preferred codon usage. These genes were cloned in a plant expression vector and then transformed into tobacco. Fifty-eight putative transgenic plants were recovered from the selected explants. Successful integration of both genes in plant genome was confirmed by PCR amplification. Expression of transgenes was confirmed by PCR amplification from total plant RNA. Detached leaf insect bioassays were conducted with Helicoverpa armigera and Spodoptera exigua larvae. About 12 % of the transgenic plants showed significantly high resistance to S. exigua. Significant mortality (62 %) of H. armigera was recorded within 24 h of bioassays. Both toxins showed synergistic effect in tobacco and broadened the spectrum of plant activity against insects.     

人干扰素α-2b 基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b（Human interferon α-2b,HuIFN α-2b）编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。     

高效烟草遗传转化体系的建立及甜蛋白基因的导入

以烟草无菌茁叶片为外植体,通过根癌农杆菌LBA4404介导法,将Thamnatin基因导人烟草中,经梯度卡那霉素(Kana-mycin,Km)筛选,获得可在含75mg／L、100mg／L Km选择生根培养基上再生的抗性植株,其中部分Km抗性植株经PCR检测为阳性,转化率为31．3％,初步鉴定已成功地建立了烟草遗传转化系统,为进一步探讨甜蛋白在植物中的转化和表达情况奠定基础。