基于PEG介导原生质体转化构建粉红聚端孢荧光标记

粉红聚端孢是多种植物的重要病原菌。本研究通过酶解粉红聚端孢幼嫩菌丝细胞壁获得原生质体,用PEG介导原生质体转化将携带GFP基因和博来霉素抗性基因的外源DNA随机插入粉红聚端孢基因组,共获得博来霉素抗性菌株90株,转化效率达18个/μg。选取22株转化子荧光观察,发现均可表达荧光,菌株间荧光强度不同,其中10株转化子荧光表达较强。与野生型相比,突变菌株TR45的菌落生长、产孢量和致病力等生物学特性均未改变,在不含博来霉素的培养皿中继代培养10代荧光仍能稳定表达。本研究构建的高效原生质体制备和PEG介导粉红聚端孢遗传转化方法,可用于该菌基因功能研究,绿色荧光标记菌株可用于病菌侵入、田间监测、侵染循环等发生规律研究。     

DNA uptake during electroporation of germinating pollen grains

Electroporation of germinating pollen grains of Nicotiana gossei (L.) Domin under a variety of conditions showed that DNA was taken up by the pollen without detrimental effects on the viability of the pollen. By optimizing both the field strength of the electroporation pulse and the DNA concentration in the electroporation medium up to 6% of the donor DNA can be taken up by the germinating pollen while maintaining a pollen viability of 90%. Field strengths as high as 9 kV/cm could be applied to germinating pollen grains without detrimental effects on viability. Southern hybridizations demonstrated that DNA encoding the marker enzyme β-glucuronidase (GUS) was incorporated into electroporated pollen. Germinating pollen, treated in this manner, is capable of producing 300–400 seeds per capsule of viable seed when applied to the stigmas of compatible flowers of N. gossei which has been emasculated 4 days earlier.     

利用有性途径的植物转化

近年来 ,植物基因工程技术取得了重要进展 ,在农作物品种改良和育种方面发挥越来越重要的作用。然而 ,目前植物遗传转化所采用的受体系统 ,大都依赖于细胞组织培养技术才能获得转基因植株。其中 ,基因型限制和遗传变异是限制该技术发展和应用的两大障碍。因此 ,一些研究者试图避开组织培养和植株再生过程 ,利用植物有性生殖途径进行转化 ,并取得了一系列成果。这些方法包括以下方面 :(1 )利用花粉粒或花粉管作为转化DNA的载体 ;(2 )将外源DNA导入子房或胚珠中 ;(3 )以精、卵细胞、合子作为转化受体。这些方法利用了高等植物的有性生殖机制和胚胎发育过程 ,避免了无性繁殖过程中的遗传变异、植株再生困难及转基因植株嵌合等问题。该文归纳综合了该研究领域所取得的成果和最新进展 ,并对这些方法进行了评价及其发展趋势进行了分析。     

Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes

The pollen tube is an excellent single-cell model system for studying cellular processes in plant cell biology. This protocol describes a detailed step-by-step procedure with optimized conditions for introducing various fluorescent reporter proteins into lily, tobacco and Arabidopsis pollen grains by means of biolistics for their transient expression and subsequent analysis in germinating pollen tubes. The whole experiment consists of four major stages: coating gold microcarriers with DNA constructs, preparation of pollen grains, transformation of plasmid DNA into pollen grains by particle delivery system and germination of bombarded pollen grains in optimized germination media to obtain pollen tubes for protein trafficking, protein localization, drug treatment and organelle dynamics analysis. This protocol takes about 4-12 h from pollen preparation to protein detection.     

激光对草原龙胆自交结实及自交后代幼苗生物量的影响

研究利用激光提高草原龙胆自交结实的方法,初步探讨了激光对花粉的生物效应。对草原龙胆的花粉进行激光照射,结果表明:适宜剂量的激光照射草原龙胆花粉,能够使其萌发率显著提高,并且使自花授粉的单朵花的结籽量也显著提高;自交后代植株的生长势有较大改善,幼苗健壮,植株的生物量也显著提高。从花粉的表面结构的电镜显微观察分析,激光照射后的花粉,其花粉壁的雕纹受到伤害,网脊线有些断裂,网眼变大,花粉管原始体变长。     

Transformation of Intact Aspergillus niger by Electroporation

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per μg of integrative vector and 100 colonies per μg of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.     

甘蓝型油菜雄性不育系160S育性转换与利用

以甘蓝型油菜雄性不育系160S为试验材料,分别在自然栽种和人工控温条件下对其花器官形态变化、花粉育性转换和杂种优势等进行了初步研究,以探讨植物温敏雄性不育的发生机制。结果表明:环境温度对160S的花器官形态及育性转换具有明显的作用,高温可使花瓣变小,雄蕊退化,花粉活力、角粒数与自交有效结角率降低,表现为低温可育、高温不育,育性变化趋势表现为完全可育-半不育-彻底败育。160S恢复源广泛,且具有较好的配合力和杂种优势,为利用两系法生产油菜杂交种提供了一个较好的途径。     

Effects of exogenous phytohormones on intracellular pH of Petunia hybrida pollen grains

The effects of exogenous phytohormones (IAA, ABA, and GA₃) on the intracellular (cytoplasmic) pH (pH_c) in ungerminating and germinating petunia (Petunia hybrida L.) pollen grains were studied. The pH_c values were measured with fluorescein diacetate. In ungerminating pollen grains, all phytohormones reduced pH_c relatively rapidly; after 10–15 min, initial value was restored. In germinating pollen grains, IAA and ABA induced a relatively rapid cytosol alkalization, which was not reversed during experiment. GA₃ acidified the cytosol, i.e., exerted the effect similar to that in ungerminating pollen grains. Sodium orthovanadate suppressed completely the hormone-induced pH_c shift toward alkaline values in germinating pollen grains, whereas this inhibitor did not affect pH_c in the absence of phytohormones. Sodium orthovanadate also slowed the recovery of pH_c after hormone-induced cytoplasm acidification in ungerminating pollen grains, reduced pH_c in control ungerminating grains, and weakened substantially the effects of all phytohormones on these pollen grains. On the basis of these results, we suggested that physiological activities of phytohormones in this system were mediated by pH_c modulation, namely, a transient disturbance in the cytosolic pH homeostasis, which could trigger further phytohormone-induced cell responses. We concluded that a hormone-induced cytoplasm alkalization in pollen grains was mediated by the activity of their plasma membrane H⁺-ATPase and that this proton pump was involved in the control of pH_c in both germinating and ungerminating pollen grains.     

外源CO及NO对干旱胁迫下水稻糊粉层细胞死亡的影响

采用荧光显微技术结合药理学方法,以水稻(Oryza sativa L.)种子及其糊粉层为实验材料,研究外源CO、NO对干旱胁迫下水稻种子萌发过程中糊粉层细胞DNA降解及死亡的影响。结果表明:(1)干旱胁迫促进糊粉层细胞的死亡,且近胚端糊粉层细胞的死亡进程早于远胚端的细胞。(2)外源CO及NO供体处理能缓解干旱胁迫下水稻糊粉层细胞DNA的降解,延迟细胞死亡进程;CO专一性抑制剂及NO清除剂能逆转CO及NO的效应,缩短细胞死亡进程。(3)外源CO及NO供体促进干旱胁迫下水稻种子的萌发,CO专一性抑制剂及NO清除剂能抑制干旱胁迫下水稻种子的萌发。(4)CO合成酶抑制剂并不能抑制外源NO对干旱胁迫伤害的缓解效应,即CO能通过NO介导调节干旱胁迫下水稻种子糊粉层细胞的死亡及种子萌发。     

Artificial Germination of Gossypium Pollen and Its utilization in Interspecific Fertilization In Vitro

The effects of different media and cultural methods on cotton pollen germination were compared. Pollen germination was successful on the improved Taylor solid medium in which the extracts of the stigmata and petals of the original species, and the comprehensive amino acids were added, respectively. The germinating pollens were viable, and active protoplasmic streaming was observed within the pollen tube. It was found that low temperature pretreatment could increase the pollen germination rate. The reasons that low temperature pretreatment stimulated pollen germination and castor oil caused abortive germination of cotton pollens were discussed. In vitro fertilization between two species of Gossypium can be promoted by smearing the stigmata with the improved Taylor medium.     

GA and ABA Regulation on the Cell Cycle in Germination of Tomato Seeds

Flow cytometric determination of ploidy levels in embryos of GA-deficient, ABA-deficient mutant and isogenic wild type tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds revealed that, large amount of 2C DNA signals existed both in wild type and GA-deficient mutant seeds, showing that most cells had arrested in the cell cycle at presynthesis Gl, whereas a relative amount of 4C proportion which is a sign of seed germination was found in ABA-deficient mutant seeds, indicating that endogenous ABA play a role in regulating the switch from development to germination in seeds. DNA replication was stimulated 1 d after the seed was imbibed in water and a visible germination occurred subsequently either in wild type GA-deficient mutant seeds. But it was not the case for ABA-deficient mutant seeds unless an exogenous GA was supplemented. This demonstrated that DNA replication in embryo root tips cells was subjected to be a compulsory factor for seed germination, whereas endogenous GA triggered DNA synthesis. It was evident that exogenous ABA could inhibit seed germination not by suppressing DNA synthesis but by bloking the route leading to mitosis since a great amount of 4C proportion was found in the germinating wild type and GA-deficient mutant seeds in the ABA solution when visible ger mination did not occur. Finally a simple mode of hormonal regulation on cell cycle in high plants was hypothesized.     

烟草脱外壁花粉的电激基因转移

以 β-葡糖苷酸酶 GUS 基因作为报告基因 ,通过瞬间表达的检测 ,比较了烟草 Nicotianatabacum L . 脱外壁花粉、未萌发与萌发花粉的电激导入效果 ,探讨了不同电激条件及启动子对外源基因瞬间表达的影响 .结果表明 :当脉冲时间常数为 13 ms时 ,导致脱外壁花粉和萌发花粉生活力下降约50 %的电场强度分别为 750 V/ cm和 12 50 V/ cm,在此条件下电激 ,二者的导入效果最好 .脱外壁花粉的GUS基因表达水平约为萌发花粉的 5倍、花粉粒的 30倍 .玉米花粉特异启动子 Zm13- 2 60 能启动 GUS基因在脱外壁花粉和萌发花粉中高效表达 ,而 Ca MV 35S的启动活性很低     

Exine-Detached Pollen of Nicotiana tabacum as an Electroporation Target for Gene Transfer

In developing alternative systems for plant transformation the authors investigated the use of male gametophyte as the foreign gene receptor. However, delivery of foreign DNA into pollen is difficult because of the existence of a thick exine, therefore a new experimental system was developed using exine-detached pollen (EDP) of Nicotiana tabacum as an electroporation target which was also compared with germinating pollen (GP) and pollen grains (P). A transient GUS expression assay was conducted to analyze the effects of different electroporation conditions and promoter activity. The pollen-specific promoter Zml3 from Zea mays mediated high level of GUS gene expression but CaMV 35S only had very low activity in both EDP and GP. The optimal field strength for gene transfer was obtained at 750 V/cm for EDP and 1250 V/cm for GP when the time constant of pulse was 13 ms. The GUS activity in EDP had a 5-fold increase as compared with GP and P respectively. The level of GUS gene expression was slightly increased when adding 10 % PEG into the electroporation buffer. This result indicates that pollen deprived of exine responds much better to foreign gene transfer than the previously used intact pollen grains and may be a better vector to introduce, via pollen tube, genes into the egg cell and offsprings.     

赤霉素与脱落酸对番茄种子萌发中细胞周期的调控

利用细胞流检仪检测番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．） ＧＡ－缺陷型、ＡＢＡ－缺陷型和相应的正常品种（野生型）成熟种子胚根尖细胞倍性水平时发现：ＧＡ－缺陷型和野生型种子绝大多数细胞ＤＮＡ 水平为２Ｃ,而ＡＢＡ－缺陷型种子则含有较多的４Ｃ细胞。在标准发芽条件下,ＡＢＡ－缺陷型和野生型种子浸种１ ｄ 后胚根尖细胞ＤＮＡ 开始复制,随后胚根突破种皮而发芽。然而ＧＡ－缺陷型种子除非加入外源ＧＡ,否则既不发生细胞ＤＮＡ 复制,也不发芽。这说明内源ＧＡ 是启动番茄种子胚根尖细胞ＤＮＡ 复制的关键因素,同时也说明番茄根尖细胞ＤＮＡ 复制是种子发芽的必要条件。实验证明：ＡＢＡ 不抑制细胞ＤＮＡ 合成,但阻止Ｇ２ 细胞进入到Ｍ 期。外源ＡＢＡ处理野生型种子与渗控处理结果相似,可以大幅度提高胚根尖４Ｃ／２Ｃ细胞的比例,但抑制种子的最终发芽     

Expression of a foreign gene in electroporated pollen grains of tobacco

Summary The incorporation of genetically engineered DNA into pollen and subsequent fertilization of eggs by the transformed pollen would be a convenient method for producing genetically engineered seed. This method of pollen transformation would circumvent the need for other types of gene transfer methods such as the use of Agrobacterium tumefaciens, which has a limited host range and thus a limited capability for genetically engineering plants. It would also avoid the problems associated with the regeneration of some plants from tissue, cell, or protoplast culture after receiving foreign DNA. To this end, the genetically engineered plasmid DNA vector pBI221 containing the gene encoding -glucuronidase (GUS) was introduced by electroporation into germinating pollen grains of tobacco (Nicotiana gossei L.). Transient expression of the GUS gene was demonstrated by the presence of GUS activity in fluorometric assays of pollen extracts 24 h after the introduction of pBI221 via electroporation. Intact pBI221 was detected by Southern blotting procedures as a distinct DNA band in pollen extracts 1 h after electroporation. In addition, pBI221 was detected as a diffuse band of higher molecular weight DNA 24 h after electroporation, suggesting that some of the pBI221 was incorporated into the genome of the pollen.     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

14种蜂花粉的DNA和RNA分析

本文使用紫外光吸收法,分析了芝麻、葵花、泡桐等１４种蜂花粉中核酸（ＤＮＡ和ＲＮＡ）的含量,以进一步探讨花粉的抗衰老作用。结果说明不论那一种花粉,均含有丰富的核酸,但种类不同的花粉其中核酸的含量是不完全相同的。     

Saving insertional recessive lethal mutants of Arabidopsis thaliana

A group of 13 recessive lethal mutants was selected on the basis of the collection of Arabidopsis thaliana transgenic plants with insertions of T-DNA vector plasmid pLD3 or pPCVRN4, which was produced by agrobacterial transformation of germinating seeds. The use of media containing exogenous hormones made it possible to compensate the lethal effect, identify phenotypes, and characterize six lines of recessive lethal germlings using genetic and molecular-genetic methods.     

氩离子注入介导麻黄基因组DNA转化获得产麻黄碱重组酵母菌

通过氩离子(Ar )注入介导蓝麻黄基因组DNA在异常汉逊酵母(Hansenula anomala)中随机转化,转化后的酵母菌经BTB指示性辅助筛选、斜面传代、液体培养、铜铬盐定性检识和RP-HPLC定量检测,获得了遗传稳定的以葡萄糖为碳源、NaNO3为氮源生物合成麻黄碱和(或)伪麻黄碱的重组酵母菌3株。液体培养72h,RP-HPLC测试胞外麻黄碱和伪麻黄碱的最高产量分别为11.87mg/L和4.11mg/L;胞内伪麻黄碱最高含量为294.86mg/g干细胞,胞内麻黄碱未检出。分析了Ar 注入介导蓝麻黄基因组DNA在酵母菌中的遗传转化效率,探讨了麻黄基因组DNA大分子的完整性对其在酵母菌中遗传转化的影响。     

Recovery of transgenic plants by pollen-mediated transformation in <Emphasis Type="Italic">Brassica juncea</Emphasis>

The aroA-M1 encoding the mutant of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) was introduced into the Brassica juncea genome by sonication-assisted, pollen-mediated transformation. The plasmid DNA and collected pollen grains were mixed in 0.3 mol/L sucrose solution and treated with mild ultrasonication. The treated pollen was then pollinated onto the oilseed stigmas after the stamens were removed artificially. Putative transgenic plants were obtained by screening germinating seeds on a medium containing glyphosate. Southern blot analysis of glyphosate-resistant plants indicated that the aroA-M1 gene had been integrated into the oilseed genome. Western blot analysis further confirmed that the EPSPS coded by aroA-M1 gene was expressed in transgenic plants. The transgenic plants exhibited increased resistance to glyphosate compared to untransformed plants. Some of those transgenic plants had considerably high resistance to glyphosate. The genetic analysis of T₁ progeny further confirmed that the inheritance of the introduced genes followed the Mendelian rules. The results indicated that foreign genes can be transferred by pollen-mediated transformation combined with mild ultrasonication.