根癌农杆菌介导Caz基因转化烟草条件研究

利用根癌农杆菌介导Caz基因转化烟草,将外源基因导入烟草愈伤组织,建成可诱导表达细胞质和细胞核CaM/CaM结合多肽的转基因烟草．研究结果表明:植物材料的选择和培养、农杆菌的培养和稀释、植物材料与农杆菌共培养的程度及转基因植株的筛选条件是影响农杆菌转化效率的重要因素．     

双价抗病基因植物表达载体构建及在烟草中表达的初步研究

多基因转化是基因工程研究热点之一。本研究应用DNA重组技术,将两个抗病机制不同,抗菌谱较广的抗病基因(天麻抗真菌蛋白GAFP和兔防御素NP1基因)构建在一个植物表达载体pBin35SGAFP-NP1上,两者具有各自的CaMV35S启动子和Nos终止子。通过根癌农杆菌介导,采用叶盘法转化烟草,PCR和PCR-Southern分析证明已将NP1和GAFP基因整合到烟草基因组中。离体抑菌实验表明转基因植株对真菌和细菌表现出一定的抗性。以上结果表明通过该表达载体进行遗传转化可获得含双价抗病基因植物,并能有效表达,提高转基因植物抗病能力。     

杨树皮储藏蛋白基因启动子的克隆和功能研究

杨树树皮储藏蛋白BSP是类似种子储藏蛋白的氮素储藏物 ,冬季在韧皮部薄壁细胞中大量积累 ,是落叶树氮代谢中的重要成分。为了研究BSP基因启动子在转基因植物中的表达特性 ,探索其在植物基因工程研究中潜在的应用价值 ,我们用PCR方法从美洲黑杨基因组中DNA扩增得到了BSA启动子片段。与GUS基因融合构建中间载体后 ,转化烟草 ,获得了一批PCR检测为阳性的转化再生植株。经GUS组织化学检测 ,发现若干转基因烟草的茎和叶柄韧皮部以及叶脉都呈GUS染色阳性 ,初步证明杨树BSP基因启动子确有韧皮部表达特性 ,可介导GUS基因在转基因烟草韧皮部特异表达。     

转录因子CBF基因转化烟草抗寒性指标的检测

为了解棉花转录因子CBF基因转化烟草的抗寒性，将苗龄21d的转基因及其野生型T2代烟苗进行0、-1、-2、-3、-4℃低温处理4h，分别进行各处理材料的电解质渗透率、丙二醛含量和可溶性糖含量的检测。结果表明转基因烟草的电导率和丙二醛含量均低于非转基因烟草，而转基因烟草的可溶性糖含量高于非转基因烟草。证实转CBF基因可以提高烟株的抗寒性。     

转基因银腺杂种杨对土壤微生物的影响

近20年来,转基因林木已开始进行田间试验,其中部分还获得商品化许可.与此同时,转基因林木的生态安全问题也逐渐受到关注,但这方面研究报道还很少.目前,已开展的转基因林木安全性研究多集中在转抗虫Bt毒蛋白基因和蛋白酶抑制剂基因林木上(高素红等,2005),而很少涉及抗逆境等其他性状基因. 土壤是生态系统中物质循环和能量转化的重要场所,转基因植物体内外源基因的表达产物有可能通过植物残留物及根系分泌物对土壤生态系统特别是对土壤微生物造成影响.评价转基因林木对土壤微生物的影响也具有重要的生态学意义(胡建军等,2004).     

植物多基因转化方法研究进展

近年来,通过转基因技术提高植物的性状、研究基因的功能获得了长足发展,不断出现的多基因转化方法为现代植物基因工程提供了更有效的手段.概述了植物多基因转化的传统方法、双元载体转化法、直接转化法和还处于起步阶段的植物人工染色体法,评论了一些方法在植物多基因转化中的优缺点,探讨了植物多基因转化中存在的植株遗传稳定性和启动子表达稳定性问题.     

Co-rrp基因小干扰RNA载体的构建及烟草中的遗传转化

为研究油茶种子成熟调控蛋白基因的功能,以pCAMBIA1304质粒为载体,根据油茶种子成熟调控蛋白基因的cDNA序列设计了小干扰RNA(Si RNA)序列,构建了油茶种子成熟调控蛋白基因小干扰RNA植物表达载体pCAMBIA1304-si RNA,并借助农杆菌介导叶盘法将pCAMBIA1304-si RNA转化到野生型烟草中,获得了稳定表达的转基因烟草苗。为下一步的油茶种子成熟调控蛋白基因的功能鉴定奠定了基础。     

马尾松PmPIN1基因的克隆及功能分析

【目的】PIN1基因通过调控生长素极性运输的方式在植物根系生长发育中发挥作用。对马尾松PmPIN1基因进行全长克隆和功能分析,有助于在分子水平揭示马尾松的生根机制。【方法】运用PCR和RACE技术成功克隆马尾松PmPIN1基因cDNA序列;利用生物信息学软件分析PmPIN1基因的核苷酸序列及其编码蛋白的氨基酸序列,并构建系统进化树;通过实时荧光定量分析PmPIN1基因在10年生马尾松幼根、1年生枝条、新叶、花中的表达量差异;利用农杆菌侵染法将PmPIN1基因转入烟草获得转基因植株,比较转基因烟草与转pCAMBIA-1302空白载体烟草之间的表型差异;用激光共聚焦显微镜观察PmPIN1-GFP荧光蛋白在转基因烟草根中的表达模式;酶联免疫法测定野生型和转基因烟草的根、根茎结合处及叶中的生长素含量;用不同浓度的生长素极性运输抑制剂1-N-萘基邻氨甲酰苯甲酸(NPA)处理野生型与转基因烟草,分析烟草根、根茎结合处、叶中的生长素含量变化;利用PEG介导法将16318-hGFP-PmPIN1重组载体转化至拟南芥原生质体中进行亚细胞定位分析。【结果】PmPIN1基因全长2914bp,包含2085bp的ORF,编码的蛋白由695个氨基酸组成,N端与C端均有膜运输蛋白。系统进化树显示马尾松与油松在发育树同一支上。通过实时荧光定量发现PmPIN1在不同组织中的表达量差异达到极显著,1年生枝条与幼根中表达量较高,花中最低。农杆菌介导法转化烟草获得的转基因烟草,较转空载烟草株高增长,生根量增加且根系变长。激光共聚焦显微镜观察得出转基因烟草根部中间有明显绿色荧光富集现象。酶联免疫法测定结果表明转基因烟草根、根茎结合处生长素含量高于野生型,且叶较根和根茎结合处减少。不同浓度NPA处理后,野生型烟草根中生长素含量变化达极显著,且生长素含量随NPA处理浓度的增加而减少;处理浓度为10nmol·L~(-1)时,叶中生长素含量最高;处理浓度为2nmol·L~(-1)时,根茎结合处生长素含量增加较多。不同浓度NPA处理后,转基因烟草不同部位生长素含量变化均未达到极显著。亚细胞定位分析表明PmPIN1蛋白主要分布于细胞膜上。【结论】马尾松PmPIN1与油松PIN1亲缘关系最近。PmPIN1属于膜蛋白。PmPIN1转基因烟草可能提高生长素由地上部位向地下部位的运输能力,减少NPA对生长素含量变化的影响,表明PmPIN1可能调控生长素的极性运输;PmPIN1-GFP绿色荧光富集在根部中间部位,PmPIN1在幼根中表达量较高,转基因烟草较转空载植株发根量多,根长长。研究结果表明PmPIN1基因可能在根系发育中发挥重要作用。     

杨树PtEBP1基因转化拟南芥研究

EBP1是一个可能在植物器官大小发育调控中发挥重要作用的基因,但其在木本植物如杨树中的功能仍未知.在前期获得杨树PtEBP1基因并构建相应表达载体pBI121-PtEBP1基础上,利用花序侵染法遗传转化拟南芥,进而通过观察转基因拟南芥的性状变化,对PtEBP1在器官发育过程中的功能进行初步探究.结果表明,杨树PtEBP1基因已成功转化拟南芥,转基因拟南芥表现为植株茎增粗、叶片明显增大且种子数量增多等显著有别于野生型的性状特征.该研究结果初步证实PtEBP1在植物器官发育,特别是器官大小发育中的重要作用.     

女贞基因转化对胁迫试剂抗性的研究

女贞（Ligustrun lucidum Ait）具有很强的防风、滞尘抗烟功能,是重要的城市绿化常绿阔叶树种之一。在山东受地域及气候的影响,该树种常常产生冻害,影响了城市园林绿化的效果。近年来,转基因技术为解决植物耐寒问题提供了有效途径。目前报道的有大豆、烟草、毛白杨等植物,通过应用转基因技术改变了某些生理特征。这些植物在转基因过程中,通常都要进行抗生素压力的确定,然后进行抗性筛选。女贞转入抗寒基因的研究尚未见报道。为将抗寒基因成功转入女贞做好准备,我们进行了女贞对协迫试剂的抗性初步研究。     

农林秸秆醋液对烤烟生长的影响

研究喷施不同浓度的农林秸秆醋液对烤烟生长的影响。结果表明：施用农林秸秆醋液后,对烟草茎粗、比叶重、叶面积有明显的促进作用,其中以农林秸秆醋液稀释300倍处理效果最好。总体说,适宜浓度的农林秸秆醋液能促进烟草的生长,在烟草生产中具有广阔的应用前景。     

植物凝集素及其在生物固氮中的作用

讨论了植物凝集素的性质、种类和分布,研究表明根瘤菌是通过对凝集素的识别来浸染植物的,因此人们提出将豆科植物的凝集素转移到其他植物中去,再接种豆科植物的根瘤菌,则可以使其他植物也被根瘤菌浸染,从而使其他植物也具有固氮的能力。     

咖啡碱合成酶基因在特异茶树资源种质创新中的利用

对咖啡碱合成酶基因在特异茶树资源种质创新中利用的基础、主要问题和前景进行了回顾和展望。对茶树中咖啡碱生物合成的了解及已有的茶树种质资源的发掘、保护和评价工作的良好基础与咖啡碱合成酶基因本身的研究成果成为咖啡碱合成酶基因在特异茶树资源种质创新中利用的基础。以茶树再生体系为重点的遗传转化体系的建立是咖啡碱合成酶基因在特异茶树资源种质创新利用中的主要问题。开发这种高科技育种技术体系，将有助于提升茶叶产业的技术水平和综合竞争力。     

利用农杆菌介导法获得转codA基因麻竹再生植株的研究

温度是影响植物生存和生长发育的基本环境因子之一。大多数植物对低温等都是高度敏感的,低温伤害现象尤为突出,几乎涉及所有的经济植物。因此,改善植物的抗低温冻害的胁迫能力,可以显著提高植物的生长范围、增加产量。codA基因可以增加植物对低温胁迫的耐受能力,而Rd29A是一种胁迫诱导特异表达启动子,胁迫条件可以快速诱导基因表达,也可以减少由于转基因过量表达带来的不利影响。研究以麻竹花药离体培养的愈伤组织为材料,采用农杆菌介导法,探讨了影响麻竹愈伤组织遗传转化效率的主要因子。结果表明,潮霉素的最佳筛选浓度是25 mg.L-1,预培养时间为3 d,侵染时间为20 min,共培养时间为3 d,乙酰丁香酮的浓度控制在100 mg.L-1时可以有效的提高遗传转化效率。在此基础上对获得的转基因植株进行分子检测,初步表明外源基因codA已经整合到麻竹基因组中。     

Species distribution models applied to plant species selection in forest restoration: are model predictions comparable to expert opinion?

An expert on local flora usually is the best option for plant species selection in most ecological restoration projects; although species selection often needs to be dealt with swiftly as well as on a limited budget, and obtaining the opinion of a local expert may not always be an economically viable alternative. In such cases, species distribution models (SDM) may offer a faster and more cost effective alternative. We asked six experts to rank native tree species according to their suitability at 24 forest sites. The predictive performance of the suitability rankings was evaluated by assessing their ability to discriminate present from absent species in the observed tree assemblages at each evaluation site. We used the area under the receiver operating characteristic curve to calculate the probability that the estimated suitability for a species present at a particular evaluation site is greater than the estimate for an absent species (both picked at random). Suitability rankings were also obtained from the predictions of SDM and the same procedure was used to estimate the predictive performance of the set of models at each site. The experts offered concordant suitability rankings at almost every evaluation site. There were no significant differences in the predictive performance of the SDM and four of the experts, although the SDM performed slightly better than the other two experts. Our results point to the suitability of the proposed species distribution modeling approach to obtain fast and cost effective recommendations for species selection in forest restoration projects.     

Smallholder perceptions of agroforestry projects in Panama

The potential effects of agroforestry systems on conservation and development have been well documented. Panama has seen a substantial rise in the number of projects with an agroforestry component in the 1990s. There has been insufficient research on the actual impacts of these projects on smallholders and of farmers' attitudes towards these systems. This study explores the perceived socioeconomic and environmental impacts of five agroforestry projects in Panama. A total of 68 smallholders were administered semi-structured interviews. In addition, 13 agroforestry experts from NGOs, government departments and research institutes were interviewed, and their responses were compared with those of the smallholders. While the projects led to an increase in the standard of living by providing wood products and fruit for domestic consumption, farm income levels generally remained unchanged. This was due primarily to limited market development, the lack of marketing organizations and poor access roads. In terms of environmental impacts, the farmers' responses suggested a slight decline in slash-and-burn agriculture, and an increase in tree planting activities. Some environmental benefits were observed by farmers, including reduced soil erosion, increased soil fertility, and improved quality and quantity of water sources. Nevertheless, slash-and-burn agriculture was still the norm for the majority of farmers. Most farmers continued to harvest wood from primary and secondary growth to meet their domestic wood requirements, rather than relying on trees planted in agroforestry projects. The main obstacles preventing increased agroforestry adoption included insufficient agroforestry extension, inappropriate project design or management (such as top-down management approaches, and the use of food incentives), smallholders' economic constraints, and larger policy issues. Recommendations are proposed to improve project design and management, and to address the economic and policy constraints. This revised version was published online in June 2006 with corrections to the Cover Date.     

山东省侧柏种质资源评价及遗传改良

山东省侧柏种质资源评价及遗传改良在国内处于领先水平,收集侧柏种源79个、家系91个、无性系26个,建立了国内首个侧柏种质资源基因库,现有侧柏采种基地2830hm2,年产优质侧柏种子200万kg。侧柏研究已经列为国家植物种质资源共享平台、国家林业局公益性行业科研专项经费项目和山东省农业良种工程课题。侧柏国家发明专利1项,审定品种6个。侧柏应将生态型(抗旱、寒)、观赏型及用材型良种选育作为为主要育种目标。本文从侧柏种质资源收集、保存及评价;良种基地建设及种子区;种源试验和种源选择;优树及家系选择;杂交育种;良种快繁技术;侧柏遗传改良策略及思路等方面对其种质及遗传改良进行了评述,以期为侧柏新种质创制奠定基础。     

Fifth International Poplar Symposium: 'Poplars and willows: from research models to multipurpose trees for a bio-based society'

Carefully managed tree plantations offer an opportunity for sustainable biomass production. In recent years, the responses of the Salicaceae to environmental constraints have increasingly been investigated at different levels of biological integration, giving rise to a physiological approach to the function of trees in environmental restoration and monitoring. Significant progress has been achieved by the poplar and willow community in understanding targeted characteristics of complex tree stress responses. The Fifth International Poplar Symposium brought together experts in this area, with the main objective being to improve, coordinate and communicate existing national research on the biological and environmental dimension of multifunctional poplar and willow plantations. The secondary objective was to develop a network of research scientists and extension workers to provide scientific support for subjects interested in using fast-growing poplar and willow species for tree-related environmental projects. The ultimate goal was to build up services for the multipurpose tree plantation network on local-level management in order to obtain maximized benefits from tree crops. The purpose was also to maximize the synergy between local knowledge and global-level processes that require information on multipurpose tree crop production.     

Defensin (TvD1) from Tephrosia villosa exhibited strong anti-insect and anti-fungal activities in transgenic tobacco plants

Plant defensin is a small, cationic, cysteine-rich broad-spectrum antimicrobial peptide with four or five disulfide bridges and has been shown to be a component of the innate immunity system in plants. In the present study, the defensin gene (TvD1) from Tephrosia villosa was overexpressed in tobacco and characterized. Two high-expression (T1, T26) and one low-expression (T13) plant lines were selected through semi-quantitative RT-PCR and used for bioassays along with non-transgenic controls. The high-expression plant line exhibited strong in vivo anti-fungal and anti-feedant activity against the pathogen Rhizoctonia solani and the first and second instar larvae of the Spodoptera litura (F.), the tobacco cutworm, respectively. The low-expression plant line showed a moderate level of tolerance/resistance in the bioassays. The recombinant peptide (rTvD1) exhibited toxicity to tobacco pollen grains in the germination assay, but transgenic plants produced copious fertile pollen and set capsules with viable seeds. The results of this study demonstrate that the single gene (TvD1) effectively controls both fungal and insect pests and, hence, it can be used for crop transformation.