Growth of Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) in Still Nutrient Medium

Cell suspension cultures were established from the callus proliferation of leaf explants of 10- to 12-day-old seedlings of the peanut (Arachis hypogaea L. var. TMV-3). The cells could be cultivated in both agitated and still media, the latter promoting more of chlorophyll (Chl) synthesis. High Chl content (210-240 micrograms Chl per gram fresh weight), yield of free and pipetable cells, presence of all the pigments in the same ratio as that of the leaf tissue, and high rates of O₂ evolution (140-170 micromoles O₂ per milligram Chl per hour) were some of the desirable features of the still-grown cell cultures. However, considerable variations with regard to the above characters were observed between the cell cultures of different varieties of the peanut.

O₂ evolution by the cultured cells was dependent on exogenous supply of HCO₃⁻. A well-developed photosynthetic apparatus as evidenced from photosystem I and photosystem II activities of the isolated chloroplasts and variable fluorescence measurements with the cell cultures was further documented by electron microscopic evidence of distinct granal stackings in chloroplasts and sodium dodecyl sulfate-polyacrylamide gel separation of thylakoid membranes into P700 Chl a protein complex and light-harvesting Chl a/b complex. Evidence is presented for the relative increase in the Chl associated with P700 Chl a protein complex in contrast to the light-harvesting Chl a/b complex in the cultured cells as compared to intact leaf.

     

Micropropagation in Peanut (Arachis hypogaea L.)

Multiple shoots in Arachis hypogaea L. could be induced from the de-embryonated cotyledons (DC), embryo-axes (EA) and mature whole seeds (MWS) in MS medium supplemented with different levels of benzylaminopurine (BAP). DC was the most suitable explant with 57.9 % induction and more than 40 shoots per explant in 31.6 % of cases. Though EA and MWS had high percent induction at or above 30 mg dm^–3 BAP, only 10 – 14 shoots per explant were observed. In DC, multiple shoots were confined to the proximal end and in EA they originated from the axillary bud region. Histological studies on DC confirmed the origin of shoots from the region of attachment with the embryo. Shoots could be rooted in MS medium containing 2 g dm^–3 charcoal and 200 mg dm^–3 casein hydrolysate. Sixty percent of the rooted plantlets could be established in the field.     

AhNCED1基因转化花生研究

构建转化AhNCED1基因花生(Arachis hypogaea L.)过表达载体35S::AhNCED1::GUS,用OD600=0.8的LBA4404农杆菌液浸染汕油523,抗性芽诱导率达100%.PCR检测89株筛选苗,43株呈阳性,GUS检测阳性率为50%.转基因植株地上部分ABA含量增加;PEG胁迫10 h,转基因植株叶片AhNCEDl蛋白表达增强,内源ABA水平积累,超氧化物水平降低.     

花生转基因研究进展

花生是世界上重要的油料和经济作物之一,是人们生活的植物脂肪和蛋白质来源。现代生物技术的不断发展为花生育种和种质创新提供了新的技术手段, 它可以直接将来自不同种属的异源目的基因插人到花生基因组, 使花生表达目标性状, 实现花生品种的遗传改良。近年来, 国内外花生转基因研究取得了重大进展。文章综述了花生转基因在抗虫、抗病、抗非生物逆境和品质改良等方面的最新进展,并总结了近年来人们对农杆菌介导法、基因枪法和不依赖组织培养的转化法等主要的花生遗传转化方法的改进和探索。     

花生根部性状的遗传分析

利用花生RIL群体,分析了11个花生根部性状的遗传力,估算基因对数及性状间的相互关系,根据偏度系数(g1)和峰度系数(g2)估算控制性状的基因互作情况。结果表明:11个性状都是受多基因控制的数量性状,在RIL群体中基因型间的差异均表现为连续变异和明显的超亲分离。侧根干重的遗传力最高达0.60,其次是侧根鲜重,为0.58,而其他性状的遗传力均较低。控制主根长性状的多基因间存在互作,互作方式为重叠作用;控制主根粗(3cm)性状的基因间也存在一定的重叠作用,但是作用不明显;控制其他性状的基因都存在互作,表现为互补作用,但互补作用的强弱有差异。主根粗(1cm)、主根粗(3cm)、主根干重、主根鲜重、侧根干重和侧根鲜重之间都显著或极显著相关;根体积与主根粗(1cm)、主根粗(3cm)、侧根干重和侧根鲜重显著或极显著相关。     

ATP Synthesis in Cell-Free Extracts of Peanut (Arachis hypogaea L.) Seeds

Cell-free extracts of peanut (Arachis hypogaea L., cv. Shulamit)seeds, incubated with various substrates, synthesized ATP. Significantsynthesis occurred in the presence of AMP + PEP, NADH₂ + PEPand NAD + PEP. When the activities were examined in extractsprepared with 0.3 M mannitol, the rates were 0.6, 0.1 and 0.04nmol min^–1 mg^–1 protein, respectively. The activitiesunder such conditions were linear with time up to 90 min incubationat 30 °C. In the presence of PEP + NADH₂ there was a higherspecific activity in extracts from non-dormant seeds than fromdormant seeds. No such difference was found when PEP + AMP orNAD + PEP was used as the substrate. The temperature dependenceof the activity showed a relatively high energy of activation(Ea) for AMP + PEP and a low one if NADH₂ + PEP or NAD + PEPwas used as substrate. In buffer extracts of seeds ATP was synthesizedin the presence of the above-mentioned substrate combinationsbut the rate of activity exhibited a lag phase at the earlytime of incubation, after which higher rates of activities (ascompared with mannitol extracts) were obtained. The activitieswere Co⁺-dependent, with a K_m of about 0.7 mM. In the bufferextracts relatively high activities of adenylate kinase (EC2.7.4.3 [EC] (AK) and pyruvate kinase (EC 2.7.1.50 [EC] ) (PK) were found.AK was stimulated by ethephon (ethylene). This effect is temperature-dependentand occurs in both directions: in the presence of ADP (ATP +AMP) as well as if ATP + AMP is used as substrate to synthesizeADP. PK is Co⁺-dependent, and unaffected by ethephon. Both activitieswere stimulated by malonate. Key words: Adenylate Kinase, Arachis hypogaea, ATP synthesis, Peanut, Pyruvate kinase, Seed     

De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons

Germination of peanut seed is accompanied by a rapid increase in isocitritase (isocitrate lyase, EC 4.1.3.1) during the first 4 days. The presence of cycloheximide (50 μg/ml) during water imbibition inhibited the increase in isocitritase activity. Actinomycin D conversely did not inhibit isocitritase activity until the second day of imbibition while RNA synthesis was inhibited. Germination of peanut seed in ¹⁴C-reconstituted amino acids followed by fractionation of a 20 to 35% ammonium sulfate preparation on a Sephadex G-200 column (57-fold purification) showed that the active enzymic fraction coincided with a large peak of radioactivity. Germination of peanut seed in 45% D₂O followed by enzyme purification and CsCl equilibrium centrifugation revealed that all the enzyme from D₂O seed had a higher density than normal isocitritase. These data indicate that isocitritase in peanut seed is synthesized de novo.     

花生LEC1基因的克隆及表达研究

通过构建花生未成熟种子cDNA文库,结合大规模EST测序,从花生中克隆了LEC1基因2个成员的全长cDNA序列.序列分析表明,花生中LEC1的2个成员的序列在B结构域高度保守,属于LEC1型HAP3亚基.不同植物中LEC1基因在B结构域以外的序列差异很大.利用花生cDNA芯片和半定量RT-PCR对花生LEC1进行表达研究表明,LEC1在种子中高水平表达,在种子发育的不同时期表达有差异.     

Reassociation Kinetics and Cytophotometric Characterization of Peanut (Arachis hypogaea L.) DNA

The base composition of peanut (var. NC-17) DNA determined from thermal denaturation profiles showed an average guanine plus cystosine content of 34% which was in close approximation to 36% guanine plus cytosine calculated from the buoyant density. Buoyant density also indicated the absence of satellite DNA. The genome size, 2.0 × 10⁹ base pairs, as determined by reassociation kinetics of the single copy DNA was close to the genome size determined by cytophotometry, 2.1 × 10⁹ base pairs. Peanut DNA averaging 450 to 600 base pairs long, reassociated in phosphate buffer and fractionated by hydroxylapatite, indicated a DNA genome composition of 36% nonrepetitive or single copy DNA; reassociation in formamide and followed by optical methods indicated the repetitive DNA possesses highly repeated, intermediately repeated and rarely repeated components of DNA with DNA sequences repeated on the average about 38,000, 6,700, and 200 times each. Different criteria of reassociation in formamide revealed further subdivisions of these four separate components of DNA. The DNA of above mentioned NC-17 variety compared to Florigiant variety showed no differences in thermal denaturation profiles, buoyant density, or in genome size.     

Endocarpic Microorganisms of Two Types of Windrow-Dried Peanut Fruit (Arachis hypogaea L.)

The endocarpic microorganisms of peanut fruit dried in either a random windrow (plants left as they fell from the digger) or an inverted windrow (plants inverted to expose fruit to sunlight) were different from that of freshly dug fruit. Chaetomium, Penicillium, Trichoderma, Rhizoctonia, and Fusarium were the dominant fungi found associated with shells (pericarp) of freshly dug fruit. The dominant fungi of shells of windrowed fruit included Chaetomium, Rhizoctonia, Fusarium, Sclerotium, and Alternaria. Seeds of freshly dug fruit were dominated by Penicillium and Aspergillus. The only dominant species in seed of windrowed fruit was Penicillium. Microorganisms were isolated from shells and seed of freshly dug fruit at a frequency of 79% and 52%, respectively. The percentage of infestation was reduced by drying in the field. This was particularly true of the inverted windrow. The proportion of shells and seed infested with a microorganism was reduced 13% and 36%, respectively, after field drying for 5 to 7 days in random and inverted windrows. Microorganisms were isolated much more frequently from shell pieces (73%) than from seed (36%).     

花生防御素基因的克隆及原核表达

定基础.     

Peanut Chlorotic Ringspot Virus (PCRV), a Newly Discovered Virus Infecting Peanut (Arachis hypogaea L.)

A virus causing wide chlorotic ringspot (PCRV) associated with chlorotic line pattern and motthng on an Erictoides hybrid growing in USDA-OSU greenhouses, Stillwater, Oklahoma, was discovered. The virus was isolated and characterized and found to differ in symptomology, host range and serological properties from all the previously described viruses infecting peanut, particularly those reported in the United States to be the most important ones, peanut mottle virus, peanut stripe virus, and tomato spotted wilt virus. The virus was transmitted by both mechanical inoctilation and grafting to fourteen peanut cultivars causing identical symptoms to those originally observed on the Erictoides hybrid. In addition to peanut, the virus systemically infected Pisum sativum L. ‘Little marvel’ causing mainly mosaic and Lupinus albus L. ‘Tiftwhite’ producing severe malformation and remarkable reduction in leaflet area. The virus did not infect many other plant species of which cowpea ‘California blackeye’ (Vigna unguiculata L.) and at least five cultivars of soybean (Glydne max L.) are known to be susceptible hosts to peanut mottle virus. Phaseolus vulgaris L. ‘Topcrop’ and Chenopodium amaranticohr Coste & Reyn were found to be two useful local lesion assay and diagnostic hosts for PCRV. The virus elicited necrotic local lesions on the first and chlorotic ringspots on the second. PCRV had a dilution end point between 10^?5 and 10^?6, thermal inactivation point between 55°C and 60°C, and longevity in vitro up to 6 days but not 7 days. Virus particles viewed hy electron microscopy and the negative stain uranyl aceute were flexuous filamentous particles ranging in length from 750–850 nm. In both indiren PAS-ELISA and Ouchterlony double immunodiffusion test, PCRV was serologically related to a PMV isolate from Oklahoma (PMV-OK.) but not to bean yellow mosaic virus, peanut stripe virus, potato virus Y, watermelon mosaic virus 1, watermelon mosaic virus 2, wheat streak mosaic virus, and zucchini yellow mosaic virus.     

花生金属硫蛋白基因AhMT3a的克隆及其表达

以鲁花14号花生为材料,从花生cDNA文库和基因组中筛选和克隆了花生的金属硫蛋白基因AhMT3a。该基因全长785 bp,有2个内含子,开放阅读框由201个碱基组成,编码66个氨基酸,其中包含13个半胱氨酸(Cys),预测其分子量为6.83kD,等电点为4.59。运用生物信息学手段对AhMT3a蛋白的信号肽、跨膜区、亚细胞定位和疏水性进行了预测。与拟南芥、棉花和草莓等植物type 3 MTs的序列比对结果表明,花生和其他不同植物的MT3在氨基酸序列上具有较高的同源性,从系统发育树中可以看出AhMT3a和蒿麦的金属硫蛋白亲缘关系较近。半定量RT-PCR和芯片杂交结果显示花生AhMT3a在花中表达量最高,在种子中表达量最低;在ABA、NaCl及PEG等不同处理下,表达量变化不大。     

花生白藜芦醇合酶基因的克隆与生物信息学分析

利用Protparam、iPSORT prediction、ProtScale、SOPMA、Swiss-Modeling和Scan Prosite等生物信息学工具分别对其理化性质、信号肽、疏水性、亲水性、二级结构和三级结构进行分析。以花生粤油45总DNA和总RNA为模板,采用PCR和RT-PCR技术克隆花生白藜芦醇合酶基因的DNA和cDNA序列,并利用SWISS-PROT、DNAMAN等生物信息学工具对其基因和蛋白质序列进行了分析。测序结果显示,该基因的DNA和cDNA序列长度分别为1 498 bp和1 251 bp,cDNA序列具有完整的开放性阅读框,编码389个氨基酸的多肽。该白藜芦醇合酶氨基酸368-378位点上存在芪合酶家族的特征位点GVLFGFGPGLT。同源性分析表明,其碱基序列与已报道的花生白藜芦醇合酶基因的一致性为99%,其氨基酸序列与已报道的花生白藜芦醇合酶氨基酸序列的一致性为100%。     

Diallel analysis in groundnut (Arachis hypogaea L.)

Summary An 8 × 8 full diallel experiment based on 4 bunch plus 4 spreading types of groundnut (Arachis hypogaea L.) was conducted over three environments. For both number of pods and pod yield, additive, nonadditive and reciprocal cross effects were detected and these were also influenced by changes in environments. For number of pods additive genetic variance was predominant whereas it was approximately equal to non-additive genetic variance for pod yield. Graphical analysis revealed the presence of strong non-allelic interaction for number of pods whereas for pod yield absence of dominance and/or presence of non-allelic interaction was evident.Part of Ph.D. Thesis of the first author     

花生叶片离体培养花器官分化及其开花结果研究

以花生幼叶为外植体进行离体培养,研究BA浓度对花器官分化的影响并进一步观察试管内花器官的发育.结果表明:经MSB 1mg/LBA 0.5mg/LKIN 2mg/LIAA培养基诱导的愈伤组织,转接到附加1～3mg/LBA的MSB培养基上培养,均能直接诱导分化花器官,但2mg/LBA的诱导效率最高达21.13%;诱导分化的花器官转接到MSB培养基继续培养,部分花器官可以在试管内开花、受精、成针、结实.试验实现了以花生幼叶为外植体,在试管内完成诱导花芽、开花、受精、形成果针、子房膨大,直至形成荚果等过程,为离体条件下研究花生花器官分化、荚果及种子发育提供了技术体系和材料.     

水培条件下花生对缺铁的生理反应

通过对5份花生(中国3份和印度2份)种质资源在水培缺铁条件下各种生理性状的研究,明确花生耐铁的生理或农艺指标,为花生耐缺铁种质资源的筛选和利用提供理论和实践依据。结果表明,缺铁明显影响品种(中花5号、远杂9102和ICG 11855)的生物产量;7d内中花8号、远杂9102和ICG 12672培养液的pH值下降3个单位,而中花5号和ICG11855仅下降2个单位。缺铁处理25d后,中花5号、远杂9102和ICG 11855幼叶的叶绿素含量、SOD、POD和CAT活性下降非常明显,而中花8号和ICG 12672幼叶的叶绿素含量、SOD、POD和CAT活性基本维持不变,这说明缺铁对中花8号和ICG 12672的叶绿素含量降低和细胞膜伤害较小。从膜氧化产物MDA的变化值也印证了缺铁对中花8号和ICG 12672的细胞膜伤害较小。综上所述,认为中花8号和ICG 12672对缺铁不敏感,而中花5号、远杂9102和ICG 11855对铁敏感,同时表明利用水培技术研究花生耐铁是一种简单有效的方法。根据相关分析,选择缺铁处理25 d后的叶绿素含量作为耐缺铁筛选的指标。     

Boron mobility in peanut (Arachis hypogaea L.)

In most plant families, boron (B) is phloem immobile. For plants such as peanut which bury their fruit, the mechanism for B delivery and the B source for fruit and seed growth remains enigmatic. Therefore, this study aimed to establish evidence of B retranslocation in peanut and to identify its importance in plant development. In a sand culture experiment, the increase in B contents in new organs after B withdrawal and the corresponding decline in B contents in older organs was evidence of B redistribution. In a foliar ¹⁰B experiment, the ¹⁰B abundance of treated-leaves decreased and ¹⁰B was detected in leaves and flowers formed after the application of foliar B. Application of ¹⁰B to the roots for a period also provided evidence for the retranslocation of ¹⁰B accumulated during the first growth period. The ¹⁰B abundance in older plant parts declined and ¹⁰B appeared in new organs (flowers, pegs, leaves) that had developed after the ¹⁰B supply had been replaced by ¹¹B. In the fourth experiment, foliar application of B reduced hollow heart, a symptom of B deficiency in seeds, in cv. TAG 24 from 39 to 8% and in Tainan 9 from 63 to 18%. These experiments all provide evidence for B retranslocation in peanut, but further work on the relative importance of the xylem and phloem pathways for B loading into the fruit is needed.