The Fertilization and Early Development of Embryo and Endosperm in Stevia rebaudiana Bertani

The mature embryo sac is surrounded by endothelium tapetum. It is composed or an egg apparatus, one central cell with secondary nucleus, and 1–6 antipodal cells. About the 6th hour after pollination, female and male nuclei fuse with each other. The syngamy occurred almost simultaneously with the fusion of an other sperm nucleus and the secondary nucleus, but the velocity of the latter is faster than that of the syngamy. The fertilization of Stevia rebaudiana Bertani belongs to the premitotic type. About the 8th hour after pollination, primary endosperm nucleus is in mitosis, its dividing orientation may parallel or at right angle to the long axis of the embryo sac, and gives rise to two initial endosperm cells. The first five divisions of the endosperm cells are of synchronism. At the stage of heart-shaped embryo, the endosperm cells show the signs of digestion and absorbed. The endosperm development is of the cellular type. About the 10th hour after pollination, zygote divides for the first time. The division of the zygote is always transverse. The embryo development conforms to the Asterad type.     

镉胁迫后旱柳转录组变化分析

随着重金属镉(Cd)应用范围的扩大,由此引发的土壤镉污染问题日益严重。以具有植物恢复潜力的旱柳Salix matsudana作为研究对象,探究不同浓度的Cd (2.5 mg/L, 50 mg/L)胁迫后旱柳无性系1 d、7 d和30 d后基因表达与代谢通路的变化。转录组测序结果表明:共获得102 595个非冗余基因(Unigenes),相同浓度不同时间的差异基因总数为26 623个和32 154个;相同时间不同浓度的差异基因总数为8 550个、3 444个和11 428个。从中筛选得到与Cd胁迫响应密切相关的基因25个,其中金属硫蛋白、ABC转运蛋白、锌和锰转运蛋白等基因的表达不仅会随着Cd胁迫浓度变化而且同时受到胁迫时间的改变而发生改变;油菜素内酯合成通路的ROT3和黄酮类化合物合成通路的FLS、F3H均明显上调。此外Cd胁迫引起旱柳在代谢过程、细胞过程、膜、细胞器、细胞、细胞部分、催化活化和结合蛋白这8个方面发生改变,参与这些GO条目的差异表达基因数随着Cd浓度和胁迫时间的增加而增加。并对转录组信息的可靠性用RT-PCR和酶活性生理实验数据进行了验证。文中通过转录组测序分析旱柳Cd胁迫后的响应机制,从而为旱柳修复土壤Cd污染提供理论指导。     

Ultrastructural Study of Mesophyll Cells During Their Dediff erentiation in Stevia rebaudiana

Authors deal with the changs of cell ultrastructure ,nucleic acid and soluble protein contents as well as peroxidase activity during mesophyll cell dedifferentiation of leaf explant of stevia rebaudiaha Bertoni. Electron microscopic observations indicated that the mature mesophyll cells had returning to meristematic cells had to go through three main phases: (1)Initiation phase :The main characteristics of cells in this phase were cytoplasmic expansion, stretching out of cytoplasmic filaments to cell centre and appearance of the protein bodies in vacuoles. (2)Medial evolving phase :The main characteristics of cells in this phase were formation of cytoplasmic bridges ;the chloroplasts were dedifferentiated to proplastids and the nuclei started to move toward centre . (3)Finishing and dividing phase :During this phase cell dedifferentiation had finished and cell division was about to start. The main characteristics of cells in this phase were the appearance of meristematic state ; the nuclei, sometimes irregularly shaped, occupied a major proportion of the cell ; the nucleoli were vaculated and the nucleopores increased in number and size. Biochemic analyses showed that the contents of the total nucleic acids , RNA and the soluble proteins as well as the peroxidase activity gradully increased during formation of meristematic cell aggregates and then reduced. However, the change of DNA contents was not obvious.     

甜菊醇糖苷生物合成关键基因的导入和鉴定分析

甜叶菊（Stevia rebaudiana Bertoni）生产的甜菊醇糖苷因具有高甜度、低热能、不参与人体内代谢兼具保健功能等特点,被誉为最有发展前途的新糖源。从甜叶菊叶片克隆了甜菊醇糖苷生物合成途径中的关键基因SrUGT85C2、SrUGT91D2m和SrUGT76G1,构建植物基因过量表达载体,以单独或组合的形式将这些基因导入到甜叶菊中,获得转基因植株。与野生型对照植株相比,单独导入SrUGT85C2的转基因植株中甜菊醇单糖苷含量提高,总糖苷、莱包迪苷A含量及占比没有明显变化;单独导入SrUGT91D2m的转基因植株中甜菊醇单糖苷含量显著降低,而甜菊醇双糖苷含量显著增加;单独导入SrUGT76G1的转基因植株中,总糖苷含量显著提高,莱包迪苷A含量达到10%以上,比对照提高了2倍,而甜菊糖苷含量减少了一半。3个基因组合同时导入的转基因甜叶菊植株与单独导入SrUGT76G1的转基因甜叶菊植株类似,其总糖苷、莱包迪苷A含量及其占比均显著提高。这些结果为以后通过分子生物学技术来调控甜菊醇糖苷生物合成关键基因的表达,培育莱包迪苷A含量高的高品质甜叶菊新品系提供了理论依据和技术方法。     

Enrichment of the rebaudioside A concentration in Stevia rebaudiana extract with cyclodextrin glycosyltransferase from Bacillus licheniformis DSM 13

Stevia rebaudiana is a sweet herbaceous perennial plant, which is frequently used in the preparation of plant-based sweeteners. The demand for such sweeteners continues to increase due to purposeful nutrition and modern-day metabolic syndromes. More than 20 types of steviol glycosides provide a sweet taste, which are more than 300 times sweeter than sucrose. They are formed of two main components, namely stevioside and rebaudioside A. Only a handful of studies have dealt with Stevia rebaudiana leaf extracts, the conversion of pure stevioside into the preferred rebaudioside A is more common. The aim of this study was to enrich the rebaudioside A content of Stevia rebaudiana leaf extract using enzymatic bioconversion by applying fermented cyclodextrin glycosyltransferase from Bacillus licheniformis DSM13. Two differently processed plant materials, namely dried and lyophilized Stevia rebaudiana plants, were extracted and compared. Following the bioconversion, the rebaudioside A content was on average doubled. The maximum increase was fivefold with a 70–80% conversion of the stevioside.     

基于转录组分析大肠杆菌响应亚碲酸盐的机制

亚碲酸盐对绝大多数微生物有高毒性,可用作抗菌剂;但其具体毒性机制仍不清楚。【目的】理解亚碲酸盐的毒性机制,揭示亚碲酸盐处理导致的代谢变化。【方法】本研究通过比较转录组分析与挖掘差异转录基因,探讨了大肠杆菌响应亚碲酸盐胁迫的机制。【结果】Escherichia coli MG1655在10μg/mL亚碲酸盐处理1 h后,比较和分析了亚碲酸盐处理组与对照组的转录水平差异,发现细胞呈现一种明显的适应性变化,许多参与重要代谢途径的基因转录水平改变。其中,与核糖体代谢和鞭毛组装相关基因的转录水平发生显著变化,表明这两条途径很可能是亚碲酸盐作用的主要途径。与细胞能动性、金属离子代谢、细胞膜功能相关的基因的转录水平也发生了明显变化,可能是由于其参与了抵抗亚碲酸盐毒性的细胞代谢调节和损伤修复。【结论】本项工作有助于推动亚碲酸盐毒性机理的研究,促进亚碲酸盐的临床应用。     

Plasmalemma Invaginations in Cultured Callus of Stevia rebaudiana: Ultrastructure and Ultracytochemical Localization of Acid Phosphatase

The plasmalemma of cells within meristematic regions was observed to possess invaginations in cultured callus of Stevia rebaudiana under differentiation. The ultrastructure and acid phosphatase (AcPase) ultracytochemistry Of these invaginations were studied. The plasmalemma invaginations occurred in the cells at various stages of vacuolation. In cells with dense protoplasm, plasmalemma appeared undulated but occasionally spherical and variable in size with conspicuous invaginations that projected into the peripheral cytoplasm. In the partially vacuolated cells, plasmalemma invagination became voluminously enlarged with increased contents and structurally complexed. In vacuolated cells, the enlarged invaginations protruded into the central vacuole but were delimitted from the tonoplast by an intermembrane zone continuous with the peripheral cytoplasm. Complex accumulations of membranes consisting of vesicular and coiled membranous Structures might develop within the plasmalemma invaginations. AcPase localization demonstrated high enzymic activity in the plasmalemma and its associated invagination. It seemed likely that these invaginations were functionally analogous to the vacuoles and therefore constituted part of the lytic compartment in these cells.     

甜菊含甙量的变异及R－A型良种的选育

从甜味成分种类及含量的不同,研究甜菊（ＳｔｅｖｉａｒｅｂａｕｄｉａｎａＢｅｒｔｏｎｉ）实生群体和单株无性系的变化,从中选育优质甜味成分含量高的良种。主要结果：（１）在实生群体中,丰产株型（圆纺锤形株）占７．３％,其中,优质甜味成分Ｒ－Ａ含量超过Ｓｔ含量的Ｒ－Ａ型株占１０．９６％,它们的Ｒ－Ａ含量变幅为３．３～１２．０％。（２）Ｒ－Ａ型良种Ｊ－２单株无性系在繁殖达２０００万株时,叶片大小和含甙量均存在极显著差异,Ｒ－Ａ含量变幅为４．５～１２．２％。（３）从Ｒ－Ａ含量为３．８６％的实生群体中选出Ｒ－Ａ含量为７．０４～１２．０３％的单株及从Ｒ－Ａ含量为９．１０％的良种单株无性系中选出Ｒ－Ａ含量为１０．１５～１２．１５％的单株,它们的Ｒ－Ａ含量均大幅度提高。     

Impact of nitrogen supply on growth,steviol glycosides and photosynthesis in Stevia rebaudiana Bertoni

This work investigated the agronomic, physiological and biochemical response of Stevia rebaudiana Bertoni grown under different nitrogen (N) rates. A pot trial in open air conditions was set up in 2012 with the aim to evaluate the effect of four N rates on the biometric and productive characteristics, steviol glycoside (SG) content as well as on leaf gas exchanges, chlorophyll fluorescence, photosynthetic pigments, Rubisco activity and N use efficiency. N deficiency caused a decrease in leaf N content, chlorophylls and photosynthetic CO₂ assimilation, resulting in a lower dry matter accumulation as well as in reduced SG production. The application of 150 kg N ha^? 1 seems to be the most effective treatment to improve rebaudioside A (Reb A) content, Reb A/stevioside ratio, photosynthetic CO₂ assimilation, stomatal conductance, N use efficiency, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) and PSII efficiency. The results demonstrate that by using an appropriate N rate it is possible to modulate the SG biosynthesis, with a significant increase in the Reb A content and in the ratio between Reb A and stevioside. This finding is of great relevance in order to obtain a raw material designed to meet consumer needs and bio-industry requirements for high-quality, Reb A content, and safe and environmentally friendly products.     

叉角厉蝽触角转录组及嗅觉相关基因分析

叉角厉蝽Eocanthecona furcellata是一种在生物防治方面有重要作用和潜力的捕食性天敌昆虫,触角是昆虫进行信息交换的重要器官,而嗅觉相关基因则是调控天敌昆虫捕食行为的重要分子基础。为获得叉角厉蝽触角转录组数据库,挖掘叉角厉蝽嗅觉相关基因,本研究利用Illumina高通量测序平台对叉角厉蝽雌雄成虫与5龄若虫触角进行转录组测序。成功构建了叉角厉蝽触角转录组,获得了67 843条unigenes, N50长度为2 300 bp。与七大公共数据库比对注释到27 686条unigenes,其中NR数据库注释最多(33.33%),且与茶翅蝽Halyomorpha halys相似度最高(64.20%)。14 258条注释到GO数据库中,分为生物过程、细胞组分和分子功能3个大类42个亚类;KEGG代谢途径分析表明,7 703条形成282条代谢通路,其中被注释在信号传导通路中的unigenes最多(11.50%)。进一步基因注释分析,鉴定得到134个候选嗅觉相关基因,32个化学感应蛋白基因(Chemosensory protein genes, CSP),10个气味结合蛋白基因(Odor...     

杜仲雄花芽2个发育时期转录组分析

杜仲（Eucommia ulmoides）雄花富含多种活性成分和营养成分,具有重要的药用和营养价值。为了揭示杜仲雄蕊原基发育相关基因的表达情况,为杜仲雄花芽发育分子调控机制研究提供理论参考。本文以杜仲良种"华仲11号"（ "Huazhong No.11" ）为材料,采用lllumina高通量测序技术,分别对苞叶原基分化期和雄蕊原基分化期的花芽进行转录组测序,通过生物信息学对2个发育时期的转录组进行比较分析,筛选出与雄花芽形态发育相关的差异基因。结果显示,转录组测序共获得40.48 Gb过滤数据,各样品的clean reads与杜仲基因组进行序列比对,比对效率为90.56%~93.01%。在2个发育时期筛选出583个差异表达基因,其中在雄蕊原基发育期上调基因315个,下调基因267个。差异基因GO和KEGG功能分析显示,差异基因富集在与生长发育、光周期途径、激素合成和信号传导、碳代谢等相关的生物过程和代谢通路。结果显示光周期途径是杜仲成花诱导的重要途径,同时雄花芽在形态分化过程中受碳水化合物、植物激素和其他代谢物质调控。此外,MADS-box家族成员FLC、SOC1、AGL3和AGL8参与杜仲雄蕊器官发育。本研究为杜仲花发育基因调控提供了基础数据,也为雄花用杜仲的分子育种提供了参考。     

暗褐网柄牛肝菌菌核形成相关基因分析

【背景】暗褐网柄牛肝菌(Phlebopus portentosus)是第一个能够人工栽培的食用牛肝菌,人工栽培过程中,不同菌株会形成数量不等的菌核。【目的】探明不同菌株产核差异机制。【方法】采集多菌核(JH1)、寡菌核(JH2)菌株的成熟菌核及无菌核(JH3)菌株培养相同时间的菌丝体进行转录组测序,分析差异表达基因对菌核形成的作用和功能。【结果】KEGG富集分析显示,JH2 vs. JH1互比,苯丙氨酸、酪氨酸和色氨酸的生物合成,精氨酸和脯氨酸代谢,半胱氨酸及蛋氨酸代谢显著富集;JH3 vs. JH1互比,乙醛酸和二羧酸代谢显著富集;JH3 vs. JH2互比,谷胱甘肽、乙醛酸和二羧酸代谢显著富集。菌核形成相关基因分析显示,从JH2 vs. JH1、JH3 vs. JH1和JH3 vs. JH2的差异表达基因中分别筛选到69、118和82条与信号转导、感知刺激、防御、碳水化合物活性酶等有关的基因,其中碳水化合物活性酶基因数量最多。三个比较组共有的碳水化合物活性酶基因在JH1中的表达量高于JH2、JH3,表明JH1更能充分利用底物营养以形成更多菌核。【结论】本研究从转录组水平初步分析了暗...     

不同施肥处理对甜菊生长及糖苷含量和积累量的影响

采用盆栽法,以甜菊( Stevia rebaudiana Bertoni)品种‘中山4号'(‘Zhongshan No.4')当年生扦插苗为研究对象,研究不同形态氮肥(硫酸铵、硝酸钠和尿素)及不同施氮量(纯氮)、施磷量( P2 O5)和施钾量( K2 O)对幼苗生长及糖苷含量和单株积累量的影响。结果显示：随氮肥、磷肥和钾肥施用量的提高,甜菊幼苗的株高、茎粗、叶长、叶宽、单株叶干质量和单株茎干质量均呈先升后降的变化趋势,且总体上与对照无显著差异,仅施磷量300 mg·kg-1处理组的叶长显著高于对照;根据施肥量与单株叶干质量的回归方程,确定硫酸铵、硝酸钠、尿素、磷肥和钾肥的施用量分别为64.87、660.21、735.84、211.54和775.92 mg·kg-1时,幼苗单株叶干质量最高。在硫酸铵处理组中,300 mg·kg-1处理组甜菊叶片中的莱鲍迪苷A( R-A)含量及甜菊苷( St)、R-A和总苷的单株积累量以及600 mg·kg-1处理组的St单株积累量高于对照,多数处理组的St、R-A和总苷含量及单株积累量均低于对照;在硝酸钠处理组中,1200 mg·kg-1处理组的R-A和总苷含量、600和900 mg·kg-1处理组的St单株积累量以及300~900 mg·kg-1处理组的R-A和总苷单株积累量高于对照,其他处理组的St、R-A和总苷含量及单株积累量均低于对照;在尿素处理组中,1500 mg·kg-1处理组的R-A和总苷的含量和单株积累量以及600和900 mg·kg-1处理组的R-A和总苷单株积累量高于对照,其他处理组的St、R-A和总苷含量及单株积累量均低于对照;各施氮处理组中,仅1500 mg·kg-1处理组的R-A含量与对照差异显著,其他指标均与对照无显著差异。在施磷处理组中,100 mg·kg-1处理组的R-A含量以及100和200 mg·kg-1处理组的St、R-A和总苷的单株积累量高于对照,多数处理组的St、R-A和总苷含量及单株积累量低于对照且与对照均无显著差异。在施钾处理组中,各处理组的St、R-A和总苷含量及单株积累量均高于对照,其中仅900 mg·kg-1处理组的St、R-A和总苷含量与对照显著差异。各施肥处理组的St含量占总苷含量的百分率均低于对照、R-A含量占总苷含量的百分率均高于对照,且总体上与对照无显著差异。经过综合分析,建议在甜菊生育期内的施肥量为纯氮600~900 mg·kg-1、P2 O5200~300 mg·kg-1和K2 O 600~900 mg·kg-1,其中氮肥以尿素为宜。     

Transcriptome analysis of differentially expressed genes and pathways associated with mitoxantrone treatment prostate cancer

The global physiological function of specifically expressed genes of mitoxantrone (MTX)‐resistant prostate cancer (PCa) is unclear. In this study, gene expression pattern from microarray data was investigated for identifying differentially expressed genes (DEGs) in MTX‐resistant PCa xenografts. Human PCa cell lines DU145 and PC3 were cultured in vitro and xenografted into severe combined immunodeficiency (SCID) mice, treated with MTX intragastrically, three times a week until all mice relapsed. Gene expression profiles of the xenografts from castrated mice were performed with Affymetrix human whole genomic oligonucleotide microarray. The Cytoscape software was used to investigate the relationship between proteins and the signalling transduction network. A total of 355 overlapping genes were differentially expressed in MTX‐resistant DU145R and PC3R xenografts. Of these, 16 genes were selected to be validated by quantitative real‐time PCR (qRT‐PCR) in these xenografts, and further tested in a set of formalin‐fixed, paraffin‐embedded and optimal cutting temperature (OCT) clinical tumour samples. Functional and pathway enrichment analyses revealed that these DEGs were closely related to cellular activity, androgen synthesis, DNA damage and repair, also involved in the ERK/MAPK, PI3K/serine‐threonine protein kinase, also known as protein kinase B, PKB (AKT) and apoptosis signalling pathways. This exploratory analysis provides information about potential candidate genes and may bring new insights into the molecular cascade involvement in MTX‐resistant PCa.