农杆菌介导的天绿香遗传转化影响因素的研究

通过农杆菌介导的方法将新疆准噶尔小胸鳖甲抗冻蛋白基因Mpafp149导入天绿香中,对影响农杆菌转化频率的各种因素进行了研究。结果表明农杆菌介导天绿香遗传转化的潮霉素最佳筛选浓度为8 mg·L^-1,预培养时间2 d,共培养时间2 d,菌液浓度OD₆₀₀为0.2～0.25,浸染时间为8 min时适宜天绿香不定芽的诱导,有利于天绿香转基因植株的获得。通过PCR检测,初步证明外源基因Mpafp149已整合到天绿香基因组中。     

不同地理种群两针松光合和生长特性的差异

为认识两针松中的赤松（Pinus densiflora）、长白松（Pinus sylvestris var. sylvestriformis）和樟子松（Pinus sylvestris var. mongolica）光合作用对环境变化的响应和适应特征,在其自然分布区内选择地理和气候差异显著的9个地理种群,采集成熟种子并播种于东北林业大学温室,2 a后,测定针叶的光合能力及其相关因子,并同时测定幼苗的株高和基径,比较种间和地理种群间差异。结果表明：赤松、长白松和樟子松种间最大光合速率（p=0.34）、呼吸速率（p=0.15）和表观量子效率（p=0.18）的差异均不显著;地理种群间表观量子效率（AQY）差异显著(p=0.08),其中兴凯湖种群表观量子效率最高,为0.084 5±0.002 4 mol CO₂·mol^-1 photons,较其他种群高13.10%~159.23%。地理种群间呼吸速率(R_d)差异显著(p=0.01),黑河和兴凯湖种群的呼吸速率最高（分别为1.62±0.18 μmol CO₂·m^-2·s^-1,1.52±0.30 μmol CO₂·m^-2·s^-1）,安图和东宁种群的呼吸速率最低,分别为0.40±0.01 μmol CO₂·m^-2·s^-1,0.34±0.03 μmol CO₂·m^-2·s^-1。地理种群间最大净光合速率(P_max)差异显著(p=0.02),其中兴凯湖、东宁、韩国、鸡东、二道白河、红花尔基种群的最大光合速率差异不显著,均值为18.36±1.81 μmol CO₂·m^-2·s^-1,高于安图、漠河、黑河种群。安图、漠河、黑河种群间最大光合速率差异不显著,均值为12.57±0.86 μmol CO₂·m^-2·s^-1。地理种群间的株高和基径差异均显著,其中韩国种群株高最高,黑河种群最低;基径兴凯湖种群最高,安图种群最低。株高和基径最大值约为最小值的3倍。两针松针叶的光合能力及其一些相关因子的地理种群间差异可能是其光合机构对种源地环境条件长期生理适应的结果。     

铝胁迫下北美车前和车前生长及叶绿素荧光特性的比较研究

采用系列浓度AlCl₃溶液（100、500、800、2 000 mg·L^-1）进行处理,在处理后不同时间（0,10、20、30 d）,测定北美车前（Plantago viriginica）和车前(P.asiatica)的生长及叶绿素荧光特征指数,旨在比较外来入侵种北美车前和同属本地种车前的生长及叶绿素荧光对铝胁迫响应的差异。结果表明,车前和北美车前的根冠比在轻度铝（Al³⁺=100 mg·L^-1）处理下略有上升,在中度（Al³⁺=500 mg·L^-1）以上铝浓度处理下开始显著下降;叶绿素a、b含量在轻度铝处理下变化不明显,随着铝浓度的增加和处理时间的延长呈现明显下降趋势;叶绿素荧光参数F_v/F_m、Φ_PSⅡ及F_v/F_o值在低浓度铝的短时间处理下略有上升,随着铝浓度的升高和处理时间的延长则明显下降。结果表明铝胁迫对上述两种植物的生长和叶绿素荧光参数均有一定影响,但北美车前各参数的下降幅度小于车前,表现出比车前更为耐铝的生理特性。     

显脉金花茶的光合生理特性研究

采用LI-6400便携式光合测定系统(Li-Cor Inc., USA)对显脉金花茶光合特性进行研究。结果表明：(1)在夏季,显脉金花茶叶片的P_n日变化呈单峰曲线,最高峰出现在中午11：00。其最大净光合速率（P_max）为3.81 μmol·m^-2·s^-1、光饱和点（LSP）为459.9 μmol·m^-2·s^-1、光补偿点（LCP）为6.9 μmol·m^-2·s^-1。显脉金花茶的光饱和点和光补偿点都比较低,表明其是一种阴生植物。（2）在控制光照强度和温度的条件下,CO₂浓度小于800 μmol·mol^-1,P_n几乎呈直线上升,升高CO₂浓度可使显脉金花茶的净高合速率增大,提高了叶片对光能的利用率。其叶片CO₂饱和点(CSP)大约在1 200 μmol·mol^-1左右,CO₂的补偿点（Г^*）为70.1 μmol·mol^-1,最大羧化速率（V_cmax）为17.5 μmol·m^-2·s^-1,最大电子传递速率（J_max）为40 μmol·m^-2·s^-1。     

浓度NO对高表达转玉米C4型pepc水稻光合的促进

为了揭示C₃植物中C₄ pepc高表达带来的生理差异与其高光合效率的关系。本文以高表达的转玉米C₄ pepc光合基因水稻（PC）及原种Kitaake(WT）为材料,通过水培在孕穗期通过根吸入的方法,进行不同浓度的NO供体、NO合成抑制剂以及相关影响信号分子的试剂单独和联合过夜处理12 h,选取倒二叶研究NO对供试材料净光合速率（P_n）,气孔导度(G_s)和胞间CO₂浓度(C_i)的影响。结果表明：WT和PC在200 μmol·L^-1 SNP（Sodium nitroprusside）和1 mmol·L^-1 L-Arg(L-Arginine)处理下,P_n分别增加20.8%、10.7%,差异显著（p<0.05）;随SNP和L-Arg浓度的增加,其表现不同程度的抑制,与PC相比,WT的P_n抑制更显著（p<0.05）,而G_s和C_i的变化则相反（p<0.05）;进一步结合200 μmol·L^-1 SNP和1 mmol·L^-1 L-Arg与SA处理,结果与高浓度的NO供体处理类似;在联合6 mmol·L^-1 Ca²⁺螯合剂EGTA处理下,与PC相比,WT的P_n抑制达到极显著水平（p<0.01）,C_i的变化则相反（p<0.05）。相关分析结果表明：PC的P_n的高低与G_s的相关性小于WT,PC与WT决定系数分别为0.654 9、0.773 5;而与C_i的相关性则更大些,PC与WT决定系数分别为0.466 5、0.419 6,显示PC可能有不同的调节方式,尤其在低浓度的NO,PC可在Ca²⁺参与下调节气孔的开放,在气孔关闭的条件下,仍能维持一定的P_n。     

影响农杆菌介导白细胞介素-2基因转化番茄的因素研究

利用农杆菌介导法将白细胞介素-2基因（il-2）导入番茄中,对影响其转化的因素进行了分析。结果表明：农杆菌菌种（EHA105和C58C1）、外植体类型（子叶和下胚轴）、带有不同筛选标记（Kan^r、PPT^r、Hyg^r）的载体质粒几个因素对芽诱导分化及转化均有影响。实验共接种转化2018个子叶和下胚轴外植体,获得了47株抗性再生株,对其进行il-2的PCR扩增检测,有44株呈阳性。PCR-Southern杂交证实PCR结果可靠,显示il-2基因已导入到番茄中。     

外源一氧化氮对铅胁迫下小麦种子萌发及幼苗生理特性的影响

利用室内水培实验,研究了外源一氧化氮（NO）供体硝普钠（SNP）对Pb²⁺处理下小麦（Triticum aestivum L.）种子萌发、幼苗生长及相关生理指标变化的影响。结果表明,Pb²⁺处理使小麦种子发芽势、发芽率、幼苗根长和茎长均显著降低,诱导叶绿素a、叶绿素b含量减少及叶绿素荧光参数F_v/F_m和F_v/F_o的比值减小,25 μmol·L^-1 SNP明显缓解Pb²⁺胁迫对种子萌发及幼苗生长的抑制作用,提高Pb²⁺胁迫下叶绿素a、叶绿素b含量及F_v/F_m和F_v/F_o的比值,而100 μmol·L^-1SNP无明显缓解作用。此外,25和100 μmol·L^-1SNP诱导Pb²⁺胁迫下小麦幼苗叶片过氧化氢酶（CAT）活性增强和可溶性蛋白含量增多,但100 μmol·L^-1SNP处理降低了过氧化物酶（POD）活性。结果说明,外源NO促进Pb²⁺胁迫下小麦种子萌发及幼苗生长,提高叶绿素和可溶性蛋白含量,诱导CAT活性升高,从而增强小麦对Pb²⁺胁迫的适应性。     

一氧化氮(NO)熏蒸蒜瓣对蒜苗叶片光合作用的影响

以白皮改良蒜为试验材料,用不同浓度的一氧化氮气体（0.1、0.5、1.0 μmol·L^-1）在无氧环境中对大蒜进行熏蒸。并使用TPS 1便携式光合仪结合Farquhar和Sharkey的理论测定或计算NO处理蒜苗的相关光合指标,同时测定核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)含量。发现与1.0 μmol·L^-1 NO气体处理相比,0.5 μmol·L^-1 NO处理的蒜苗叶片净光合速率（P_n）、气孔导度(G_s)提高、而胞间隙CO₂浓度（C_i）、气孔限制值(L_s)降低,说明0.5 μmol·L^-1 NO处理下蒜苗光合速率高于1.0 μmol·L^-1 NO的处理的主要是非气孔因素。而且0.5 μmol·L^-1 NO处理提高了蒜苗叶片表观量子效率(AQY)、表观羧化效率(CE)和光合能力(A_o)及Rubisco含量,说明外源NO处理提高了蒜苗叶片光合作用过程中光反应能力和碳同化过程中羧化酶羧化效率。与对照相比,1.0 μmol·L^-1 NO处理降低了蒜苗叶片净光合速率,同时气孔导度、胞间隙CO₂浓度、表观量子效率、Rubisco含量、羧化效率和光合能力均降低,而气孔限制值升高,说明1.0 μmol·L^-1 NO对蒜苗光合作用的抑制既有气孔因素,也有非气孔因素。而0.1 μmol·L^-1 NO处理各项指标与对照无显著性的差异。     

外源一氧化氮对NaCl胁迫下黄瓜幼苗生长、活性氧代谢和光合特性的影响

采用营养液水培的方法,研究了外源一氧化氮（Nitric oxide, NO）对50mmol•L^-1NaCl胁迫下黄瓜幼苗生长、活性氧代谢和光合特性的影响。结果表明：10~400μmol•L^-1 NO供体硝普钠（Sodium nitroprusside, SNP）能显著缓解NaCl胁迫对黄瓜植株造成的伤害,100μmol•L^-1 SNP缓解效果最好,可提高幼苗的生长量,增强幼苗叶片超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）、抗坏血酸过氧化物酶（APX）活性,提高了叶片叶绿素和脯氨酸（Pro）含量、净光合速率（Pn）、蒸腾速率（Tr）及气孔导度（Gs）;降低了叶片丙二醛（MDA）和过氧化氢（H₂O₂）的含量、超氧阴离子（O^•-₂）的产生速率、质膜透性和胞间二氧化碳浓度（Ci）。     

Ri质粒介导的几丁质酶基因转化小麦的研究

采用L₉（3⁴）的正交试验,以带有pMLH7133-Chi质粒的发根农杆菌为介导,对栽培品种春小麦东农7742、龙麦9814和Hite的幼胚愈伤组织进行了遗传转化研究。结果表明：基因型为东农7742、菌液浓度OD₆₀₀=0.8、共培养时间2 d、乙酰丁香酮（As）浓度为100 μm是最佳的试验组合;各影响因素的主次顺序是：基因型＞乙酰丁香酮的浓度＞菌液浓度＞共培养时间。共获得138株Hyg抗性植株,经PCR及PCR-Southern检测初步证明,外源几丁质酶基因已整合到其中5株的基因组中,又对3株阳性植株进行了RT-PCR扩增和几丁质酶活力测定,证明外源几丁质酶基因已在小麦基因组中稳定表达。     

Overexpression of MinE gene affects the plastid division in cassava

The MinE protein plays an important role in plastid division. In this study, the MinE gene was isolated from the cassava (Manihot esculenta Crantz) genome. We isolated high quality and quantity protoplasts and succeed in performing the transient expression of the GFP-fused Manihot esculenta MinE (MeMinE) protein in cassava mesophyll protoplasts. The transient expression of MeMinE-GFP in cassava protoplasts showed that the MeMinE protein was located in the chloroplast. Due to the abnormal division of chloroplasts, overexpression of MeMinE proteins in cassava mesophyll protoplasts could result in fewer and smaller chloroplasts. Overexpression of MeMinE proteins also showed abnormal cell division characteristics and minicell occurrence in Escherichia coli caused by aberrant septation events in the cell poles.     

根癌农杆菌介导的苜蓿体胚转化

以苜蓿体细胞胚胎作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究该转化体系的最佳实验参数。实验结果显示,负压处理10min和共培养5d时表达率最高(可达17．4％)。以这一转化方法分别对带有3种不同启动于的表达载体进行比较,发现由CMV35S启动于驱动的GUS基因的瞬时表达率可达82．7％,Ubil启动于驱动的可达57．8％,而Actl启动于驱动的则未见表达。     

Production of stably transformed cassava plants via particle bombardment

 A novel protocol, based on biolistics and regeneration via organogenesis, was developed for genetic transformation of cassava (Manihot esculenta Crantz). The in vitro performance of cassava cultivars CMC40, MPer183 and MCol22 was evaluated, and the regeneration protocol was modified to improve shoot production from explants for transformation experiments. Somatic cotyledons were used as a target tissue in the transformation experiments using the Particle Inflow Gun and a plasmid containing the uidA gene in transient assays. The effect of different parameters for particle bombardment efficiency, including the amount of DNA used, the flying distance of the projectiles and the pre- and post-plasmolysis time of the target tissue, was evaluated and the conditions were partially optimised. Stably transformed cassava plants of cvs. MCol22 and TMS60444 were produced using the partially optimised conditions and two different vector constructs carrying the hpt gene as the selectable marker. The selection protocol was optimised further, and a rooting test was developed for screening the regenerants for antibiotic resistance to reduce the number of escapes obtained after primary selection. The production of stably transformed cassava lines and the expression of the transgenes was verified by Southern blot analysis and RT-PCR. Received: 10 December 1999 / Revision received: 12 January 2000 / Accepted: 7 February 2000     

影响农杆菌介导的木薯基因转化因素的研究

本文研究了影响农杆菌介导的木薯基因转化的因素。结果表明,供试的4个菌种中,LBA4404(pBin9GusInt)及LBA4404(pTOK)瞬时表达效果较好。对农杆菌的诱导处理能增强瞬时表达效果,外植体的预处理对瞬时表达无影响,而外植体的预培养显著降低瞬时表达。所有供试的木薯品种都能被农杆菌侵染,但外植体的类型及生理状况对农杆菌的侵染力影响很大,成熟胚状体的子叶(萌发15d)及试管苗完全展开的叶片对农杆菌亲和性最高。四种筛选剂(kanamycin、hygromycin、phosphinothricin及geneticin)均表现出剂量效应且能同步抑制芽器官发生、愈伤生长及芽切段生根。     

影响农杆菌介导的木薯基因转化因素的研究

Factors influencing agrobacterium-mediated cassava transformation were investigated. Among the four Agrobacterium strains tested, LBA 4404 (pTOK 233) and LBA 4404 (pBin9GusInt) gave higher transient expression than C 58 C1 (pIG121Hm) and EHA 105 (pBin9Husint). Pretreatment of explants by bombardment or vaccum had no significant effect on transient expression while preinduction of Agrobacterium with acetosyringone showed better effects, and preculture of explants showed worse effects. All the cultivars tested were susceptible to Agrobacterium infection, while the types of explants and the physiological state of the explants had a strong influence on the transient expression efficiency. The 15-day-old somatic cotyledons and the fully expanded leaves from in vitro plantlets were the most susceptible to Agrobacterium infection. The results also showed that all the four selective reagents (hygromycin, geneticin, PPT, and kanamycin) synchronously suppressed the growth of callus, shoot organogenesis and shoot rooting in a dose dependent manner.     

不同转化条件对3种农杆菌GFP基因在本氏烟草中瞬时表达的影响

以本氏烟草（Nicotiana benthamiana）为植物材料,分析了不同农杆菌菌株（LBA4404菌株、EHA105菌株、GV3101菌株）、菌液浓度以及侵染时间在瞬时转化过程中对报告基因GFP荧光表达量的影响。结果显示,不同的农杆菌菌株瞬时表达外源基因的最适浓度和时间均有所不同：LBA4404菌株在菌悬液OD₆₀₀值为0.8时所介导的瞬时表达效率最高;而EHA105和GV3101菌株在菌悬液OD₆₀₀值为0.6时可达到最高瞬时表达效率。LBA4404菌株所介导的瞬时表达在农杆菌注射后第2天时表达量最高,而EHA105和GV3101菌株所介导的瞬时表达在农杆菌注射后第4天时表达量最高。不同菌株间比较分析表明,LBA4404菌株所介导的瞬时表达效率最高。上述结果表明,农杆菌菌株以及浓度和侵染时间等转化条件均是影响瞬时表达效率的重要因素。     

A Versatile Agrobacterium-Mediated Transient Gene Expression System for Herbaceous Plants and Trees

Plant transient expression is a powerful method used widely for the functional characterization of genes and protein production. In comparison with stable transformation, it has the advantages of being simple, quick, economical, and effective. In the present study, we developed a novel transient gene expression system based on Agrobacterium-mediated transformation. This system is simple and convenient and allows for high transient expression levels. Hyperosmotic pretreatment of plants significantly improved the transient expression in this system. Furthermore, other factors, including acetosyringone concentration, cocultivation time, and Agrobacterium cell density, significantly influenced transient expression efficiency. The results showed that this method is suitable for use with herbaceous plants (such as tobacco and Arabidopsis) and trees (such as birch, poplar, tamarisk, cork, willow, and aralia), suggesting that it may be applied widely in plant transient expression studies.     

Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz)

In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.     

抗氧化剂对农杆菌介导的大豆下胚轴GUS基因瞬时表达的影响

大豆(Glycine max)下胚轴作为大豆遗传转化的外植体材料,能快速高频再生不定芽。然而,在遗传转化过程中褐化影响基因转化效率。在该研究中,我们用含有GUS染色基因和hpt II(Hygromycin phosphotransferase II)筛选基因的农杆菌(Agrobacterium tumefaciens) LBA4404侵染大豆下胚轴,并用组织化学定位法测定了GUS基因的瞬时表达,以确定大豆的优化基因转化条件。结果显示,在共培养基中加入硫代硫酸钠、L_半胱氨酸以及二硫苏糖醇等抗氧化剂,可以有效地抑制大豆下胚轴在组培过程中褐化的发生,并大幅度提高农杆菌在下胚轴的瞬时表达率。这些结果说明抗氧化剂可以降低这种影响并有效提高基因转化效率。     

农杆菌介导巨桉Eg5高效遗传转化

以巨桉(Eucalyptus grandis)无性系Eg5叶片为外植体, 探讨了农杆菌(Agrobacterium tumefaciens)侵染时间、共培养pH值和共培养时间对瞬时转化效率的影响, 分析了不同筛选策略对遗传转化植株筛选效果的影响。结果表明, 外植体侵染45分钟, 共培养pH值为5.8, 共培养3天所得到的瞬时转化效率最高; 逐步提高卡那霉素(Km)浓度筛选转基因植株有效, 筛选率达到15%, 转化率达到0.26%。经过GUS染色分析和PCR检测, 证实为转基因植株。