甜蛋白基因MBLⅡ对番茄的遗传转化

以5～7d龄的“丽春”番茄无菌苗子叶作为外植体,研究了子叶外植体对抗生素卡那霉素的敏感性,抗生素卡那霉素对番茄筛选的适宜浓度为70mg／L。通过根癌农杆菌(Agrobacterium tumefaciens)介导,成功地进行了马槟榔甜蛋白基因MBLⅡ对番茄的遗传转化,获得转化番茄抗性植株,组织化学法有阳性表现、PCR特异扩增及Southern杂交检测出现特异条带,表明MBLⅡ基因已顺利整合到转基因番茄植株的基因组。     

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。     

甜蛋白Brazzein基因在番茄果实中的特异表达

西瓜(Citrullus vulgaris S.)来源的AGPL1启动子在番茄(Lycopersicon esculentum L.)果实中具有较强的特异性驱动功能。将该启动子与甜味蛋白基因Brazzein融合构建植物表达载体, 通过根癌农杆菌(Agrobacterium tumefaciens)介导法成功地进行了对番茄的遗传转化, 获得转化植株。组织化学法、PCR特异扩增、Southern杂交分析及RT-PCR检测, 表明Brazzein基因已整合到转基因番茄植株基因组中并且稳定表达。通过AGPL1果实特异启动子的调控, 在不改变果实其他性状的前提下提高了番茄果实甜味品质, 并为甜蛋白的生产提供经验。     

胸腺肽基因对樱桃番茄的遗传转化

胸腺肽基因是一343bp的小肽基因,是从动物中克隆得到的。本文以“美味樱桃”番茄为植物材料,用农杆菌侵染法进行了胸腺肽基因的遗传转化。对所得再生植株进行了PCR和Southern blot检测及RT-PCR检测,34棵Kam抗性株通过目的基因PCR检测,4棵为阳性株,阳性株率为11．8%。实验中还对目的基因之后的Nos终止序列区进行了扩增,通过Nos Ter．引物对4株目的基因PCR阳性株作PCR检测．只有1株为阳性株,该植株经Southern blot检测和RT-PCR检测,均为阳性。这些检测结果说明胸腺肽基因成功地整合到番茄基因组中,并在转录水平上得以表达。     

天花粉蛋白基因转化番茄的研究

核糖体失活蛋白（ｒｉｂｏｓｏｍｅｉｎａｃｔｉｖａｔｉｎｇｐｒｏｔｅｉｎ,ＲＩＰ）是一类作用于核糖体,抑制蛋白质合成的蛋白毒素,它具有广谱抗植物病毒和动物病毒的活性,在异种植物中的抗性效应尤其明显。迄今为止,人们已经从５０多种植物中分离出６０多种不...     

花青素调节基因VlmybA2对番茄遗传转化

以小型番茄 Micro-Tom 为材料,利用农杆菌介导法导入花青素调节基因VlmybA2。对抗性筛选出的再生植株进行 GUS 组织染色和 PCR 检测,证明外源基因已经整合到 Micro-Tom 中,转基因番茄根、茎、叶脉、果皮均呈紫色,花色为黄紫嵌合。而野生型的根为白色,茎、叶脉呈绿色,果皮为红色,花为黄色。对转基因番茄的花青素含量、叶片叶绿素含量和光合速率等生理指标进行测定,花青素含量有显著增加,叶绿素含量降低,VlmybA2基因过量表达会降低植株的光合效率,但对植株正常生长影响并不显著。VlmybA2 基因既可增加抗衰老物质花青素含量,又可作为转基因植株的报告基因。     

高效马铃薯遗传转化体系的建立及甜蛋白基因的导入

本研究选用了三个马铃薯(Solanum Iuberosum L.)栽培品种“85T-14-3”、“86-2”及“Favorita”的块茎、微型薯和试管薯为起始材料,应用根癌农杆菌 Ti 质粒系统成功地建立了一种方法简单、速度快和频率高的遗传转化体系。其中试管薯薄片的转化速度最快,经根癌农杆菌(Agrobacterium tumefaciens)共培养后,薄片在100mg/L 卡那霉素的分化培养上,2—3周就可产生出抗性小芽,这些小芽进一步仲长后可在50—100mg/L 卡那霉素的无激素MS 培养基上生根。从共培养到转化植株的获得只需6—7周。微型薯和试管薯的转化频率较高,最高可达67.5%。大多数抗性植株均能检测到胭脂碱合成酶或 GUS 基因的表达。把带有甜蛋白基因和胭脂碱合成酶标记基因的Ti质粒导入马铃薯,获得大量转化植株。叶片抗性检测和 nopaline 检测可推断外源甜蛋白基因已进入马铃薯基因组。     

大叶补血草质膜Na~+/H~+逆向转运蛋白基因SOS1的克隆及对番茄的遗传转化

根据大叶补血草(Limonium gmelinii(Wildl.)Kuntze)SOS1基因序列(登录号:EU780458)设计特异性引物,通过RT-PCR方法从叶片中克隆得到质膜Na+/H+逆向转运蛋白基因LgSOS1,将该基因重组于质粒pCAMBIA1390的CaMV35S启动子下游,构建含LgSOS1基因植物表达载体pCAMBIA1390-LgSOS1。通过根癌农杆菌介导法转化番茄(Lycopersicon esculen-tum),在1.2 mg/L6-BA、0.2 mg/L IAA、20 mg/L潮霉素和200 mg/L羧苄青霉素的MS培养基上进行选择培养,从抗性愈伤组织中获得再生植株。经PCR和RT-PCR检测,初步证实LgSOS1基因已整合至番茄的基因组中。     

SARS S1基因对番茄的遗传转化与转基因番茄植株的获得

利用DNA直接导入法将含有SARS S1基因的植物表达载体pCS1导入农杆菌LBA4404中,并通过农杆菌介导的叶盘转化法得到12株再生小苗。PCR、Southern杂交检测结果证实,有6株为真正的转基因植株。     

绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .     

A simple method for in planta tomato transformation by inoculating floral buds with a sticky Agrobacterium tumefaciens suspension

Tomato transformation is conventionally performed using Agrobacterium tumefaciens-infected cotyledons. Here, we propose a simple procedure for tomato transformation, by which A. tumefaciens cells were smeared onto floral buds of a tomato plant using a paintbrush. Sufficient numbers of fruits were obtained from them, although the smearing of an excess number of A. tumefaciens cells led to an adverse effect on the plant growth. Progeny plants were screened by growth on a kanamycin-containing selection medium plate. The nptII gene was detected in 10 plants among 1,599 progenies. These transformants were derived from fruits other than those obtained from the smeared buds. This suggested that A. tumefaciens cells moved to the buds located near the smeared buds and caused the transformation event. Our findings suggest that this procedure can be used for the introduction of a foreign gene into plant cells.     

Distribution and diversity of begomoviruses in tomato and sweet pepper plants in Costa Rica

Begomoviruses (genus Begomovirus, family Geminiviridae) have emerged as important plant pathogens in tropical and subtropical regions worldwide. Although these viruses were reported during the 1970s in Costa Rica, they are still poorly known. Therefore, the objective of this study was to analyse the diversity and distribution of begomoviruses in commercial tomato and sweet pepper fields from different agricultural production systems of the major growing regions of Costa Rica. A total of 651 plants were randomly sampled from greenhouses and open field crops during 2011 and 2012 in three different geographical locations. The bipartite begomoviruses Tomato yellow mottle virus, Tomato leaf curl Sinaloa virus and Pepper golden mosaic virus, and the monopartite begomovirus Tomato yellow leaf curl virus were detected in the collected samples. The complete genome of isolates from each species was cloned and sequenced. The frequency of detection of these four begomoviruses in the analysed samples ranged from 0 to 9%, the presence, and the prevalent virus varied largely according to the geographical location, the host (tomato and pepper), and the production system (greenhouses or open fields). An association between geographical region and begomovirus species was observed suggesting that in Costa Rica the heterogeneity on climate, topography and agricultural system might influence the distribution of begomovirus species in the country. A broader survey needs to be conducted to confirm it, although these preliminary results may contribute to the management of begomoviruses in Costa Rica.     

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。     

番茄热激蛋白90的全基因组鉴定及分析

热激蛋白90(Heat shock protein 90,Hsp90)是植物应对不良环境胁迫产生的一类特定的抗逆蛋白。文章以番茄(Solanum lycopersicum L.)基因组数据为平台,借助生物信息学方法对Hsp90基因家族进行鉴定与分析。结果表明,番茄至少含有7个Hsp90基因,不均匀分布在6条染色体上,氨基酸序列长度为267~794aa,内含子数目为2~19;共线性分析发现两对基因(Hsp90-1和Hsp90-3,Hsp90-5和Hsp90-7)以片段重复形式存在。MEME(Multiple Em for Motif Elicitation)分析显示,番茄Hsp90基因编码的氨基酸序列具有多个保守基序;聚类分析揭示番茄、水稻(Oryza sativa L.)和拟南芥(Arabidopsis thaliana L.)Hsp90基因可以分为5组,存在3对直系同源基因和4对旁系同源基因;基于RNA-seq数据库表达分析发现,3个基因(Hsp90-5、Hsp90-6和Hsp90-7)在营养器官和生殖器官中表达量较高,4个基因(Hsp90-1、Hsp90-2、Hsp90-3和Hsp90-4)除在番茄转色后10 d的果实中表达量较高外,其余组织中表达量均较低;对Hsp90基因启动子序列进行分析,发现了多个参与植物对逆境胁迫的顺式作用元件,如HSE、CCAAT-box。此外,qRT-PCR检测结果表明,在叶片热胁迫条件下,番茄Hsp90基因的表达量均存在增强趋势,表明这些基因参与了番茄叶片应对高温胁迫的反应。研究结果为鉴定番茄Hsp90基因的功能和进化起源奠定了基础。     

不同来源甜高粱种质资源的表型遗传多样性分析

通过对主要农艺性状的方差分析及聚类分析,对194份不同来源的甜高粱种质资源进行了表型性状的遗传多样性分析。结果表明,不同来源品种间的性状差异较大,与中国品种相比,国外品种具有植株高大、生物产量高及含糖量高等优异性状,可用于国内资源的种质创新及品种改良。遗传距离为0.66时将所有资源划分为6类,各类群主要按农艺性状进行了划分。研究结果将为杂交育种的亲本选择提供理论参考。     

雪花莲外源凝集素基因转化番茄

将含有雪花莲外源凝集素基因（ＧＮＡ）的质粒ｐＲＳＳＧＮＡ１通过冻融法转化到根癌土壤杆菌９Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）菌株ＬＢＡ４４０４中。采用叶盘法转化番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．）栽培品种“Ｃ８”、“Ａ３９”和“Ａ５３”,获得了含ＧＮＡ基因的４３株转化植物株。通过卡那霉素抗性鉴定、ＮＰＴⅡ基     

An experimental assessment of the factors influencing Agrobacterium-mediated transformation in tomato

Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pTOK233, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency evaluated by a GUS gene transient expression level. Eight factors including explant types, explant size and source, the concentration of cytokinin, inoculation time, pH of inoculation and cocultivation media, bacterial concentration, acetosyringone concentration, and cocultivation duration were investigated in detail. This optimized protocol was then adopted to obtain transgenic tomato plants resistant to cucumber mosaic virus (CMV) mediated by Agrobacterium tumefaciens, strain LBA4404, carrying a binary vector pR-ΔGDD containing the kanamy cin-resistance gene and CMV replicase gene with GDD deletion. The presence of the CMV-RNA2 gene was confirmed by genomic DNA Southern blot analysis in all transformants analyzed. Field spray test showed that the transgenic tomato plants were resistant to 100 mg/l kanamycin. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284. The text was submitted by the authors in English.     

Chemical synthesis and characterization of the sweet protein mabinlin II

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste. © 1998 John Wiley & Sons, Inc. Biopoly 46: 215–223, 1998     

雪花莲外源凝集素基因转化番茄

The plasmid pRSSGNA1 carried a snowdrop lectin gene (Galanthus nivalis agglutinin, GNA) under the drive of RSs-1 promoter, were successfully transferred into three tomato (Lycopersicon esculentum Mill.) cultivars, “C8”,“A39” and “A53” via Agrobacterium-mediated transformation. Regenerated plantlets were obtained from cotyledons after preculture, shoot inducing culture and root inducing culture. Transgenic tomato plants were confirmed by the kanamycin-resistant experiment, PCR analysis and Southern blot. The preliminary results from bioassay demonstrated significant resistance of the transgenic plants to aphid (Myzus persicae Sulzer) larvae. The inheritance of selective marker gene (NPTⅡ) in 3 transgenic tomato plants is in the model of the simple Mendel's fashion in progenies of the selfing generation.