Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

ABSTRACT: BACKGROUND: Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to only few plant species and often requires substantial equipment and facilities. The high content of chloroplasts and chlorophyll can impede downstream applications of transformed cells from green plant tissue. RESULTS: We describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima) for which no particular growth facilities or expensive equipment are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in all commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to a low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Transgene expression is detectable within 90 min post-transformation and lasts over several days. CONCLUSIONS: The simplicity of isolation and transformation renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multicolour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.     

草莓果实外源基因瞬时表达系统的建立

将东北红豆杉紫杉烷13α-羟基化酶(Taxane13α-hydroxylase,13OH)基因全长cDNA正向插入到pCAMBI-A1304,构建其植物表达载体pCT13OH;将水稻小RNA169d(microRNA169d,miRNA169d)基因前体(precursor)全长DNA正向插入到pCAMBIA1305.1,构建其植物表达载体pCO169d。用电击法把它们导入根癌农杆菌GV3101,获得有关工程菌株。用1mL无菌注射器分别将这2种工程农杆菌悬浮液注射到草莓(Fragaria×ananassa)授粉后1周、2周、3周的果实中,发现授粉后2周果实适宜注射,可用于瞬时表达。对授粉后2周的果实进行不同注射部位、根癌农杆菌不同活化时期和根癌农杆菌悬浮液不同注射量试验,以蒂部注射、根癌农杆菌活化12h和0.5mL悬浮液注射量的效果最好,与结构基因13OH和调节基因miRNA169d相融合的GUS基因都得到表达,瞬时表达成功。     

一种植物表达载体的构建及其在金柑上的瞬时表达

以植物表达载体pCambia1301的带内含子的GUS(iGUS)基因替换植物表达载体pBI121中的GUS基因,构建了以pBI121为基础载体并含有iGUS的植物表达载体pLZ13。农杆菌染色表明,pCamiba1301和pLZ13两载体中的iGUS基因均不会导致GUS染色反应。以金柑不同组织为材料,通过农杆菌介导开展瞬时表达研究,结果表明,pLZ13和pCambia1301两载体的iGUS在CaMV35S启动子调控下的表达没有差异,证明构建的pLZ13是一种同时适于瞬时表达和转基因研究的植物表达载体。     

龙眼DlLac7的克隆及其表达调控分析

【目的】探索漆酶(laccase)与龙眼生长发育及逆境胁迫响应之间的关系。【方法】根据龙眼胚性愈伤组织转录组数据库unigene序列,利用RT-PCR和RACE技术,以‘红核子’龙眼胚性愈伤组织的c DNA为模板,克隆laccase基因,并利用实时荧光定量PCR技术对其在龙眼体胚发生过程中、不同组织部位和逆境胁迫响应中的表达进行分析。【结果】获得龙眼漆酶laccase基因2个转录本Dl Lac7-a和Dl Lac7-b的c DNA全长序列(KM103385和KM103386)。Dl Lac7-a和Dl Lac7-b全长分别为2 121 bp和2 064 bp,包含相同的完整开放阅读框(ORF)1 713 bp,均编码570个氨基酸,2者仅3’非编码区(3’UTR)长度不同;该氨基酸序列与甜橙、毛果杨和葡萄等物种的laccase有较高的同源性。生物信息学分析表明,Dl Lac7的保守结构域具有漆酶的典型结构域特征。q PCR分析结果表明,在龙眼体胚发生过程中,Dl Lac7在鱼雷形胚时期的表达量最高,子叶胚阶段下调表达,其他阶段表达量平稳;Dl Lac7在龙眼成花中表达量最高,其次是花芽,在根中也有一定程度的表达,而在其他组织部位表达量较低。激素和非生物胁迫处理下的表达分析表明,水杨酸(SA)、低浓度茉莉酸甲酯(Me JA)、盐、渗透、干旱和脱落酸(ABA)胁迫等因素均可诱导Dl Lac7上调表达。【结论】Dl Lac7可能参与龙眼体胚发生中期的转录调控过程,且可能参与茉莉酸、SA和ABA的逆境胁迫信号转导途径,调控龙眼多种非生物胁迫应答过程。     

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background  

Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.     

梨组织培养及遗传转化研究进展

组织培养及遗传转化体系的构建对梨体细胞变异筛选和品种改良研究具有重要意义,从外植体选择、培养基配方、培养条件3个方面对梨组培快繁体系构建进行了综述,重点阐述了遗传转化外源基因类型、外植体和菌株选择、培养以及检测方法,指出了现阶段梨再生及遗传转化体系存在的问题,并对未来的研究重点进行了展望,以期为今后梨砧木及品种改良相关研究提供参考。     

Estimates of genetic and environmental variability of biochemical constituents and colour in processing-tomatoes in Southern Nigeria

Seven cultivars of tomato (Lycopersicon esculeutum Mill.) were planted at 2 locations and 2 seasons to obtain estimates of their genotypic and environmental variability. pH, titratable acidity, reducing sugars, total soluble solids and pulp colour were affected by season, while the titratable acidity and colour were also affected by location. All cultivars were found suitable for processing with regard to pH, but the other characteristics varied with cultivar, some being below standards for processing in some cultivars. The characteristics were heritable to an appreciable degree, indicating that a new adapted cultivar may be developed.     

In vitro callus-induction in globe artichoke (Cynara cardunculus L. var. scolymus) as a system for the production of caffeoylquinic acids

Summary

Globe artichoke (Cynara cardunculus L. var. scolymus) provides a rich dietary source of bio-active compounds derived from phenylpropanoid metabolism, notably caffeoylquinic acids (CQAs) and flavonoids. Micropropagation techniques have been established for this species, but in vitro cultures have not yet been extended to generate an efficient system for the induction of callus tissue. In this study, we compared more than 100 combinations of media supplements (e.g., phytohormones, absorbers of polyphenols, and inhibitors of polyphenol oxidase), along with various light regimes, and three different genotypes of globe artichoke to define the optimal conditions for callus induction from leaf explants. This led to the elaboration of an in vitro culture protocol which resulted in a high frequency of callus induction after just 1 week in culture. The procedure used leaf explants from virus-free, meristem culturederived plantlets. Quantitative HPLC analysis revealed that, as in globe artichoke leaves, the predominant phenolic esters present in callus were mono- and di-caffeoylquinic acids (diCQA). The concentration of diCQA was three- to five-fold higher in calli than in leaves. The exposure of calli to UV-C light further enhanced the levels of CQAs. In vitro callus culture combined with UV-C irradiation may thus represent a viable production system for diCQA that is suitable for the synthesis of pharmacologically-active compounds.     

African nightshades: genetic,biochemical and metabolite diversity of an underutilised indigenous leafy vegetable and its potential for plant breeding

African nightshades are becoming more important as leafy vegetables in sub-Saharan Africa. Previously considered as food for the poor, their cultivation is now being promoted, and some cultivars are commercialised; however, most farmers use self-produced seeds, leading to low and varying yields. Improvement through conventional breeding depends on the available genetic diversity, the possible breeding systems, and the nutritional value of the accessions. Therefore, we review the information on these topics with the following main outcomes: the most commonly discussed species, S. nigrum, S. scabrum, S. villosum, and S. americanum, could be differentiated using molecular markers, but further sub-clustering was rarely possible, and statistical support often missing. S. nigrum and S. scabrum seem to be most closely related to each other. The mainly self-pollinating African nightshades form a polyploidy series with diploid (2n = 2x = 24) to hexaploid taxa. Interploidy hybridisations between diploids and tetraploids are possible, whereas the hexaploid S. nigrum and S. scabrum could not be crossed to genotypes of lower ploidies. Solanine, solamargine, solasonine, and chaconine are the major steroidal alkaloid glucosides in African nightshades. Amounts are age and environment dependant. Mineral and vitamin contents in leaves are at least as high as in Brassica oleracea or Spinacia oleracea, underlining their relevance as local vegetables.     

不同铁肥品种对苹果叶片叶绿素、铁含量及光合速率的影响

采用断根输液法对缺铁失绿症的苹果树分别施用不同浓度的柠檬酸铁、螯合铁和邻啡罗啉铁3种铁肥,试验结果表明,断根输液能在较短时间内提高叶片的叶绿素和铁的含量,提高叶片的光合速率,从而获得较好的复绿效果。邻啡罗啉铁是适合根系输液矫正苹果树缺铁失绿症的铁肥品种。     

柑橘冷诱导基因及其启动子表达载体构建与瞬时表达分析

【目的】以YFP为报告基因,构建柑橘冷诱导基因及其启动子的植物表达载体。【方法】克隆获得枳冷诱导基因Ptcor8及其启动子p Ptcor8,柠檬中同源基因Clcor8及其启动子p Clcor8;双酶切含启动子的克隆载体和植物表达原始载体p1D4,连接获得含启动子的中间载体;再双酶切含冷诱导基因的克隆载体和中间载体,连接后获得重组质粒;通过冻融法将重组质粒导入农杆菌EHA105中。【结果】构建了p1D4/p Ptcor8-Ptcor8::YFP,p1D4/p Ptcor8-Clcor8::YFP和p1D4/p Clcor8-Ptcor8::YFP 3个融合表达载体,瞬时表达发现3个融合基因均可在冰糖橙叶片中表达。【结论】3个融合表达载体的成功构建为下一步转化柠檬,分析枳冷诱导基因Ptcor8及其启动子p Ptcor8的功能奠定了基础。     

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.     

Biolistic transformation of broccoli (Brassica oleracea var. italica) for transient expression of the b-glucuronidase gene

Summary

We report for the first time conditions for the biolistic transformation of broccoli. Efficient transient expression of the b-glucuronidase (gus) gene has been obtained following transformation of broccoli cv. Marathon F1 (Brassica oleracea var. italica) cotyledons via microparticle bombardment. The influence of several particle gun delivery parameters and pre-treatment of cotyledon leaf discs on rates of transient GUS expression have been investigated. Consistently high rates of transformation were obtained using M17 tungsten particles fired at 900 psi with a gap distance of 20 mm and a target distance of 55 mm. Bombardment of cotyledon leaf discs placed on callus induction media doubled rates of transient transformation events. Pre-culturing cotyledon leaf discs on either hormone-free media or callus induction media resulted in a decrease in transient transformation events.     

Optimizing shoot regeneration and transient expression factors for Agrobacterium tumefaciens transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency

An efficient, adventitious shoot regeneration protocol was devised, and transient expression studies were carried out to enable Agrobacterium-mediated stable transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency. Leaves, from in vitro stock cultures, with the petiole removed and four partial cuts made transversely and equidistant through the midrib area were found to be the optimum explant type. A 24 h liquid TDZ-pretreatment (0.05, 0.10 or 0.25 mg/l) in MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497.] of leaf explants stimulated shoot formation upon subsequent culture on QL medium [Quoirin, M., Lepoivre, P., 1977. Improved media for in vitro culture of Prunus sp. Acta Hort. 78, 437–442.] supplemented with 3.0 mg/l BAP and 0.5 mg/l NAA. A frequency of 38.9–54.4% of the explants produced at least one shoot with the maximum mean number of shoots, 4.5 per explant with the 0.10 mg/l TDZ pretreatment. The shoot regeneration scheme was subsequently linked with inoculation with Agrobacterium tumefaciens strains EHA105, GV3101 or LBA4404, each harboring the binary plasmid pBISN1. PBISN1 contains an intron interrupted ß-glucuronidase (GUS) gene (gusA) under control of the chimeric super promoter (Aocs)₃AmasPmas. Blue stained leaf cells were observed after co-cultivation with all three strains. Co-cultivation for 4 days with 19.6 mg/l acetosyringone (AS) and assay by GUS indicated over 90% of the leaf explants were infected with an average 7.5–8.8 blue foci per explant. No differences were observed in regard to A. tumefaciens strain used.     

Relationship between genetic polymorphisms of GSTM1 as well as GSTT1 and lung cancer

AIM and METHODS:To examine the relationship of glutathione S-transferase M1(GSTM1) and T1(GSTT1) with the occurrence of lung cancer, The case-control study was conducted among 161 lung cancer and 165 healthy controls. The genetic polymorphisms of GSTM1 and GSTT1 were detected with the method of multiplex polymerase chain reaction. Logistic regression analyses were used to assess the interaction of between different genotypes as well as between null genotypes and smoking. RESULTS:The frequences of GSTM1 and GSTT1 null genotypes had no obvious difference between lung cancer and healthy controls. In non-smoking subjects, the frequence of GSTM1 null genotype was significantly different between lung cancer and healthy controls. Furthermore, GSTM1 null genotype was significantly overrepresented in adenocarcinoma patients aged 60 or over, compared with controls.The results from interaction analyses showed although smoking and GSTM1 deletion were associated with an increased risk of lung cancer, GSTM1 and GSTT1 null genotypes combined with smoking did not have interaction effect on the risk of lung cancer. The risk for adnocarcinoma in the individuals at the age of 60 or over and in nonsmokers without GSTM1 gene but with GSTT1 functional genotype decreased by 48.5% and 45.3%, respectively. CONCLUSION:Our results suggest that GSTM1 deletion is an important host risk for lung cancer, and imply that GSTT1 functional genotype protects the old (aged 60 or over) and nonsmokers who are lack of GSTM1 gene from the risk of adenocarcinoma.