矮牵牛花器官发育的研究进展

介绍了矮牵牛花器官发育基因的克隆、表达及其在花器官发育中的作用。     

一种新型矮牵牛花发育突变体的研究

报道了新发现的一种矮牵牛(Petunia hybrida L.)花发育突变体,命名为efficient(eff),并对这一突变体进行了形态学和遗传学分析。eff突变体主要表现为雌蕊心皮数目的增加和雄蕊上长出花瓣状结构,同时,雄蕊、花瓣和萼片数亦有增多,但营养器官无变化。心皮数目的增加导致雌蕊柱头和子房体积的显著增大,并形成较大的果实。雄蕊上花瓣的形成对花粉的产生无明显影响。扫描电镜观察发现,eff突变体在花器官原基形成时发生了相应数目的增加及特征的变化。遗传学分析表明,突变的表现型符合孟德尔单基因遗传规律。     

同源四倍体矮牵牛花粉母细胞减数分裂观察

以同源四倍体矮牵牛06P-12为材料,采用常规压片法对花粉母细胞减数分裂过程及染色体行为进行了观察研究,以探明同源四倍体矮牵生育性降低的细胞学原因.结果显示:花粉母细胞减数分裂过程与二倍体基本相同但有其特殊性,主要表现在:终变期染色体的构型复杂,有四价体、三价体和单价体;中期Ⅰ和中期Ⅱ有赤道板外染色体;后期Ⅰ和后期Ⅱ出现落后染色体、丢失染色体、染色体桥及不均等分裂的现象;四分体时期出现一分体、二分休、三分体以及含微核的异常三分体、四分体、多分体.花粉母细胞减数分裂过程中正常细胞平均达78.6%,异常细胞频率平均为21.4%.研究表明,同源四倍体矮牵生育性降低的细胞学原因是减数分裂过程中染色体行为异常.     

矮牵牛花药培养及植株再生研究

采用花粉发育双核期的矮牵牛花药,研究不同浓度植物生长调节剂配比及不同浓度蔗糖和麦芽糖对花药诱导率的影响。结果表明,采用6-BA和IBA组合诱导效果较好;NH+6-BA 1.5mg/L+IBA 1.5mg/L花药诱导率较高;麦芽糖诱导效果明显比蔗糖好。     

观赏植物花色基因工程研究进展

花色是观赏植物的重要性状,创造新花色是花卉育种的主要目标之一。基因工程技术 在观赏植物花色育种上可弥补传统育种技术的缺陷,因此它在花色育种方面的研究和应用发 展迅速。本文从花的成色作用和花色素种类入手,介绍了花色苷的生物合成,并从花色基因 的种类和克隆、花色基因工程操作的策略和方法等角度综述了近年来观赏植物花色基因工程 的研究进展。同时对我国观赏植物花色基因工程的前景作一展望。     

矮牵牛花同源异型基因fbp2的克隆及其对烟草花形态的影响

以矮牵牛（Ｐｅｔｕｎｉａ　ｈｙｂｒｉｄａ　Ｌ．）栽培品种为材料,取开放前的花蕾分离ｍＲＮＡ,反转录合成ｃＤＮＡ,以ｃＤＮＡ为模板,通过ＰＣＲ扩增,对获得的目的片段进行序列分析。结果表明,分离的目的片段含有６８６个核苷酸（含有起始密码和终止密码）。核苷酸序列与文献报道相比,同源率为９９．６％,只有３个碱基发生改变,５’端的ＭＡＤＳ盒区域完全相同。将得到的矮牵牛花同源异型基因ｆｂｐ２的ｃＤＮＡ（ｙｆｂｐ２）与ＣａＭＶ３５５启动子和ＮＯＳ３’终止子融合,构建了表达载体ｐＢＢＰ２。表达载体通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）ＬＢＡ４４０４（ｐＡＬ４４０４）介导转化烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．）叶片,在含有１００ｍｇ／Ｌ卡那霉素的抗性培养基上再生成株。对抗性株进行总ＤＮＡＳｏｕｔｈｅｒｎ杂交和总ＲＮＡ的点杂交,证明目的基因已导入烟草细胞中,整合到烟草基因组上,并且在烟草细胞中转录。同源异型基因ｆｂｐ２导入烟草后导致烟草花型改变,在雄蕊上产生了花瓣。     

葡萄基因工程研究进展

植物基因工程技术为培育优良葡萄品种开辟了一条全新而有效的途径。葡萄基因转化受体系统的建立主要包括器官发生途径和胚状体发生途径,建立良好的受体系统是葡萄基因转化成功的关键,遗传转化途径主要有根癌农杆菌介导的遗传转化和基因枪法。概述了迄今国内外葡萄基因工程的研究进展,着重对葡萄基因转化受体系统的建立、转化的方法、转化植株的筛选和检测、影响葡萄基因转化的主要因素等进行了综述,并展望了葡萄基因工程的发展前景。     

观赏植物花色基因工程研究进展

花色是观赏植物的重要性状,创造新花色是花卉育种的主要目标之一。基因工程技术在观赏植物花色育种上可弥补传统育种技术的缺陷,因此它在花色育种方面的研究和应用发展迅速。本文从花的成色作用和花色素种类人手,介绍了花色苷的生物合成,并从花色基因的种类和克隆、花色基因工程操作的策略和方法等角度综述了近年来观赏植物花色基因工程的研究进展。同时对我国观赏植物花色基因工程的前景作一展望。     

二倍体矮牵牛花粉母细胞异常减数分裂的观察与核型分析

以夏季高温时期二倍体矮牵牛花蕾为材料,采用常规制片法,对花粉母细胞异常减数分裂进行观察并选取终变期进行染色体核型分析。结果发现:异常减数分裂主要表现为具有多核仁、二价体提前分离成单价体、赤道板外的染色体,姊妹染色单体提前分离、不均等分离,落后和丢失染色体,具有微核的三分体和四分体,中期Ⅱ纺锤体定位发生异常出现融合纺锤体和八字形纺锤体可导致2n花粉产生;矮牵牛终变期核型公式为K(2n)=2x=14=10m+4sm(2SAT),其中第1、4、5、6、7号为中部着丝粒染色体,第2号(具有随体)、3号为亚中部着丝粒染色体,染色体相对长度组成为2n=14=4M_1+10M_2,核型分类为2A型。研究表明,矮牵牛异常减数分裂可导致2n花粉和不育花粉的产生,利用终变期进行核型分析具有材料丰富、二价体形态清晰不需人为配对分析等优点,为矮牵牛细胞学研究提供了新方法。     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

香雪兰花瓣的花色苷组成

为研究不同品种香雪兰的花色苷组成、含量及与花色表型之间的关系,阐明香雪兰花色形成机理,该研究以不同花色的香雪兰(Freesia hybrida) 11个品种为材料,采用英国皇家园艺学会比色卡(RHSCC)和色差仪进行花色描述,利用特征颜色反应初步确定色素类型,通过pH示差法测定花瓣中总花色苷的含量,进而利用UPLC-Q-TOF-MS技术分析各品种花瓣中花色苷种类和相对含量。结果表明:11个所选品种涵盖香雪兰四大色系,即白色系、黄色系、红色系、蓝紫色系;所选品种都含有黄酮类化合物,不含或含有极低量的类胡萝卜素,除‘White River’‘Fragrant Sunburst’‘Gold River’‘Tweety’外,均含有花色苷;‘Red Passion’花瓣中总花色苷含量最高,最低是‘Lovely Lavender’,其含量仅为‘Red Passion’的24%;在香雪兰花瓣中共检测出10个花色苷组分,分别为飞燕草-二葡萄糖苷、矢车菊素-二葡萄糖苷、矮牵牛素-二葡萄糖苷、飞燕草素-3-O-葡萄糖苷、矢车菊素-3-O-葡萄糖苷、芍药素-二葡萄糖苷、锦葵素-二葡萄糖苷、矮牵牛素-3-O-...     

矮牵牛中查尔酮合成酶基因A (chsA) 2个启动子的克隆和分析

查尔酮合成酶(chalcone synthase,CHS)是类黄酮类物质生物合成途径中的第一个关键酶,其控制基因chs为超家族基因.根据前人报道的矮牵牛查尔酮合成酶基因A(chsA)启动子的保守序列设计1对特异性引物,从矮牵牛基因组DNA中通过1次PCR同时扩增出长为550 bp和354 bp的启动子(分别命名为PchsA-L和PchsA-S,GenBank登录号:EF199747和EF199748),其中PchsA-L与PchsA-S相比,除个别碱基有差异外,在88～269 bp多出一段182 bp的序列,其中103～201 bp含有典型的内含子特征.应用DNAStar软件分析表明2条序列均含有普通启动子的保守序列TATA box、CCAAT box、capsite(CCATAA),并含有花中特异表达启动子的特征序列TACPyAT box、anther box(TAGAAGTGACAGAAAT)、G-box(CACGTG)、box1元件(ATGTCACGTGCCATC)和box2元件(TGTGTTGAAGGTTTGCTA).对克隆启动子所用的矮牵牛后代群体进行分析,130个单株中只含有PchsA-S的有13株,只含有PchsA-L的有20株,同时含有2个启动子的有97株.2个启动子在后代中发生了分离,但其分离比并不符合1∶2∶1.克隆启动子所用的矮牵牛有14条染色体,为二倍体.DNA印迹表明2个启动子在基因组中均是多拷贝.qRT-PCR分析显示:未经过紫外光处理的花中以及经过紫外光处理的花中,PchsA-L启动子驱动的chsA基因与PchsA-S启动子驱动的chsA基因表达都未见明显差异;在紫外光处理的幼苗叶片中表达量相应地比紫外光处理的花中的表达量增高;在紫外光处理的幼苗叶片中,PchsA-L启动子驱动的chsA基因比PchsA-S启动子驱动的chsA基因表达量显著增高;而未经过紫外光处理的幼苗叶片中,PchsA-L启动子驱动的chsA基因、PchsA-S启动子驱动的chsA基因都没有检测到明显的表达信号.结果表明:在矮牵牛中chsA基因存在2个独立的启动子PchsA-L和PchsA-S;启动子PchsA-L中182 bp类内含子特征的序列具有显著提高chsA基因在紫外光处理的幼苗叶片中表达量的功能.     

Enhanced frequency of transposition of the maize transposable elementActivator following excision from T-DNA inPetunia hybrida

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assayAc activity inPetunia hybrida. In other species, such as tobacco orArabidopsis, excision ofAc from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion ofAc was consistent with a low primary transposition frequency (0%–0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%–17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies ofAc. No evidence was found for an altered methylation state forAc following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.     

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Summary Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.     

溶磷真菌ATMT转化条件优化及其对矮牵牛的促生效果评价

[目的]建立和优化一株溶磷真菌咖啡果小蠹青霉菌Penicillium brocae的转化体系,并利用分子标记观察其在根部的定殖;检测接种咖啡果小蠹青霉菌对植物的促生作用,为菌肥的研发奠定基础.[方法]利用农杆菌介导的转化体系(Agrobacterium tumefaciens-mediated transformati...     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

施用甲醇刺激矮牵牛的生长效应与其代谢关系初探

大量研究表明施用甲醇能够促进多种植物的生长,在甲醇刺激植物生长的机理中,支持碳源假说的证据最多。该研究通过考察矮牵牛甲醇代谢与甲醇刺激其生长的相关性,对碳源假说进行验证。结果表明:(1)在MS固体培养基上添加2和6mmol/L CH3OH均可促进矮牵牛的生长和叶绿素含量增加,但2mmol/L CH3OH(低浓度)效果好于6mmol/L(高浓度),而且添加6mmol/L CH3OH会诱发较强的氧化胁迫。(2)进一步用13 C-NMR分析矮牵牛对不同浓度13 CH3OH的代谢作用发现,6mmol/L 13 CH3OH处理矮牵牛中[U-13 C]Fruc和[U-13 C]Gluc的生成量显著大于2mmol/L 13 CH3OH处理,即来自甲醇的碳源在代谢过程中虽被卡尔文循环同化为糖类物质,但这部分碳源对甲醇刺激矮牵牛的生长贡献不大。这些证据表明CH3OH代谢与其刺激矮牵牛生长的效果没有关联性,该实验结果不支持碳源假说。     

遮荫对月季光合特性及生长发育的影响

采用人工遮荫法研究不同程度遮荫(75%、50%和25%全光照,以全光照为对照)处理对月季的生长发育、光合特性及叶绿素荧光参数的影响。结果表明:(1)随着遮荫程度的增加,月季的叶片厚度、花径、成花率均呈降低趋势,叶绿素含量呈增加趋势;净光合速率(Pn)、蒸腾速率(Tr)、气孔导度(Gs)、胞间CO2浓度(Ci)降低,气孔限制值(Ls)升高,Pn下降的主要原因可能为气孔限制。(2)月季在遮荫条件下的光饱和点和光补偿点、暗呼吸速率下降,表现出其对光照降低的生理适应机制;其光饱和点在600~1 200μmol·m-2·s-1,光补偿点在29.89~62.95μmol·m-2·s-1,CO2补偿点在78.16~89.41μmol·mol-1,CO2饱和点在1 100μmol·mol-1左右,潜在光合能力13.06~25.63μmol·m-2·s-1,其对光的适应范围较宽,光照和CO2浓度对其光合能力的影响较大。(3)各处理条件下叶绿素荧光参数显示,月季在自然光和低光下(50%和25%全光照)叶片PSⅡ受到了明显伤害,光合作用原初反应过程受到抑制,光合电子传递受到影响。研究发现,月季虽是阳生植物,但光照生态幅较宽,轻度遮荫(75%全光照)能促进其光合作用效率,减少光抑制,植株长势好,成花率高。