Analysis of a Lysozyme Gene from Sweet Potato Hornworm, Agrius convolvuli

ABSTRACT ABSTRACT Lysozyme, the bacteriolytic enzyme, is one of the factors of the innate immunity in insects. A cDNA sequence encoding a lysozyme was isolated from the fat bodies of sweet potato hornworm, Agrius convolvuli immunized with E. coli K12 D21. The coding region of this lysozyme consists of a 19 residues signal peptide and a 120 residues mature protein, which is very similar to that of other lepidopteran. Thirteen negatively (Asp and Glu) and twenty two positively (Arg, Lys and His) charged amino acids were found in this sequence. Eight Cys residues were conserved in the same positions of other insect lysozymes.     

Isolation of Lysozyme from the Hemolymph of Sweet Potato Hornworm, Agrius convolvuli Larvae

ABSTRACT Lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. A new family member of insect lysozyme, an antibacterial peptide, has been isolated from the hemolymph of the fifth instar larvae of Agrius convolvuli . Vaccination was proceeded with E. coli K12 D21 (4x10⁶ cells of log phase) injection into the abdomen of the larvae. Agrius lysozyme was isolated by cation-exchange chromatography and RP-FPLC, and sequenced by HPLC system. It was observed that the purified Agrius lysozyme was heat-stable and had a molecular weight of approximately 15 kDa by SDS-PAGE. The N-terminal amino acid sequence of Agrius lysozyme is K-H-F-S-R-C-G-L-V-Q-E-L-F-W-Q-G-F-P with the highest similarity to that of Heliothis virescence , and it has been identified that the Agrius lysozyme belongs to c type lysozyme.     

Neuronal Cell Death of Abdominal Ganglia (A3) and Characterization of Neuron-Killing Factor in Ventral Nerve Cord from Sweet Potato Hornworm, Agrius convolvuli

ABSTRACT The central nervous system of Agrius convolvuli is composed of brain, followed by subesophageal ganglion, three thoracic ganglia, and eight abdominal ganglia in late larval stage. After metamorphic transition from larva to pupa, thoracic (T₁ and T₂) and abdominal ganglia (A₁ and A₂) are moved toward T₃ and fused together to construct composite ganglion, pterothoracic ganglion. The formation of composite ganglion is completed about 90% at 4 day and 100% at 7 day after pupation. Neuronal cell death was occurred significantly around 3 or 4 day after pupation and just after adult ecdysis. Although 170 neurons were detected 3 day before adult ecdysis, 24 cells were counted 5 day after adult ecdysis. Data of scanning and tandem electron microscope showed the symptom of cell death. In order to identify the mechanism of cell death in A. convolvuli , 1,200 ventral nerve cords were homogenized. Extracts were boiled for 3 minutes at 100°C and collected below 30,000 dalton of molecular mass. Each fraction from reverse phase column chromatography by HPLC system was tested in ventral nerve cord culture system, and fractions having killing activity in culture were isolated. By the addition of 20 hydroxyecdysone, actinomycin D, or cycloheximide into the culture medium, cell death was delayed significantly when compared to control group.     

黑化诱导激素对白薯天蛾的发育影响

黑化诱导激素是一种神经肽,它像激素一样调控昆虫的生长发育。给五龄白薯天蛾注射较高浓度的黑化诱导激素时,它们的吐丝期明显延长,蛹重也明显增加。给五龄各天白薯天蛾注射136pmol每克体重的黑化诱导激素,它们的添食期、吐丝期延长,蛹重增加。因此,黑化诱导激素会延长天蛾幼虫的添食期和吐丝期,影响天蛾幼虫的发育。     

Localization of Allatostatin-Producing Cells in Larval Gut of the Potato Worm Agrius convolvuli

ABSTRACT This study has been carried out to investigate localization of allatostatin-producing cells in the gut of Agrius convolvuli using an immunocytochemical method. Allatostatin producing cells could not be found in the foregut and hindgut, but they were abundantly distributed only in the posterior part of midgut. These endocrine cells had typically columnar shape, with secretory surface positioned to muscular layer of midgut wall and also showed different intensity of immunoreactivity to AST, mainly with strong or moderate intensity.     

Hypoglycemic and hypolipidemic properties of oil derived from Agrius convolvuli

This study aims to determine the hypoglycemic and hypolipidemic properties of oil derived from Agrius convolvuli (Lepidoptera: Sphingidae) in KKAy mice, a cross between diabetic (KK) and lethal yellow (Ay) mice. A group of 12 KKAy mice were fed with AIN-76 feed containing oil derived from A. convolvuli pupae (ACPO) while another group of 12 KKAy mice were fed with AIN-76 feed without ACPO, for 6 weeks. Compared with the control group, the KKAy mice fed with the ACPO diet had decreased serum insulin, glycogen, fasting blood glucose, triacylglycerol, total cholesterol and low-density lipoprotein cholesterol level, but had increased serum high-density lipoprotein cholesterol level, linolenic acid content and total polyunsaturated fatty acid content. The results indicate that ACPO has hypoglycemic and hypolipidemic properties and may be a suitable alternative food supplement for humans.     

甘薯天蛾过冷却点变化与生化成分的关系

测定长沙地区甘薯天蛾Agrius convolvuli L.在发育过程中不同时期的过冷却点、虫体水分和粗脂肪含量,并对其动态变化和相互之间的相关性进行分析。结果表明:不同世代甘薯天蛾过冷却点均随发育进程显著升高,在蛹期明显下降;2龄幼虫的过冷却点均为-11℃左右,蛹的过冷却点在越冬代过冷却点为-7·38℃,比其他世代蛹低2~3℃。甘薯天蛾虫体含水量随发育进程不断降低,粗脂肪含量则不断升高,其越冬代蛹粗脂肪含量占其干重高达30·23%。甘薯天蛾过冷却点与虫体水分含量呈正相关;而与虫体粗脂肪含量呈负相关,且在第2、3代和越冬代均呈显著负相关。甘薯天蛾在发育过程中通过调节水分和粗脂肪含量可能是其抵抗低温的生理机制之一。     

Cecropin D‐like antibacterial peptides from the sphingid moth,Agrius convolvuli

Two major antibacterial peptides were isolated and purified from immunized larval hemolymph of Agrius convolvuli. Acid extraction, gel filtration, ultrafiltration, and reversed‐phase FPLC were used for purification of peptides. These peptides had similar molecular mass and amino acid composition. Moreover, 21 of the first 23 N terminal residues were identical. The peptides were highly homologous with cecropin D in size and primary sequence, and named Agrius cecropin D1 and D2. The molecular masses of Agrius cecropin D1 and D2 were 3,879.39 and 3,839.27, respectively. In antibacterial and hemolytic assays, Agrius cecropin D showed potent antibacterial activities against a panel of Gram positive and negative bacteria without hemolytic activity against human red blood cells. Notably, our antibacterial assay revealed Agrius cecropin D possessed stronger or at least equivalent activities against B. megaterium than cecropin A. It suggests that Agrius cecropin D, which has an alternative structure from cecropin D, could be the model for the development of peptide antibiotics. Arch. Insect Biochem. Physiol. 41:178–185, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of an Arylphorin-type Storage Protein in the Sweet Potato Hornworm, Agrius convolvuli

A major hemolymph protein (M_r 480,000) in the larvae of the sweet potato hornworm, Agrius convolvuli, was purified and characterized. This protein was isolated with a high yield from the hemolymph of day 3 fifth final instar larvae by ammonium sulfate precipitation and Phenyl-Sepharose and Q-Sepharose column chromatographies. The protein has two subunits, an M_r 84,000 subunit (α) and an M_r 80,000 subunit (β), and the native protein was composed of a heterohexamer (α₃β₃). The two subunits have similar amino acid compositions, with high contents of aromatic amino acids (about 15% phenylalanine plus tyrosine) and low levels of methionine. The N-terminal amino acid sequences of both subunits showed high homologies with insect arylphorin-type storage proteins. The protein concentration in the hemolymph increased steeply from day 3 final instar larva and reached a maximum level of 42 mg/ml in females and 41 mg/ml in males among wandering larvae. The concentration in the hemolymph declined once during the larval–pupal transformation but remained high during the early–mid pupal period and almost disappeared after adult emergence. These quantitative changes were the same for males and females. Based on these characteristics, we identified the hemolymph protein as an arylphorin-type storage protein.     

甘薯天蛾幼虫体色分化动态的数值化评价指标及其应用

为解决无法用文字实现对近千种连续变化的昆虫体色进行准确的描述和合理的数学运算问题, 本文采用国际标准颜色计量技术-PFG (Pantone Formula Guide) 颜色指南为昆虫体色性状判别标准, 提出了关于数值化描述昆虫体色的相关评价指标, 如体色元 (body-color element, BCE) 、 体色群 (body-color group, BCG) 、 体色位 (body-color position, BCP)、 高频色码指数 (high frequency color-code index, HFI) 和杂色指数 (variegated color index, VCI) 等及其定量分析方法。并应用这些指标和方法, 研究了甘薯天蛾Agrius convolvuli幼虫个体在相似环境条件下体色分化的多样性以及不同发育日龄和不同虫体部位（体位）其体色的演化过程及其数值规律, 发现甘薯天蛾幼虫存在着共同的主体色, 而且其体色的变异主要发生在个体的不同发育日龄（占总变异的72.66%）, 其次, 虫体不同部位的颜色也有明显的差异（占总变异的24.45%）, 而同一发育日龄个体间的体色差异仅占总变异的2.88%。 结果说明, 甘薯天蛾幼虫体色随其发育日龄所发生的变化过程, 可能是由遗传因子所决定的一种生命体从发育到衰老的表观呈现过程。     

湖南甘薯种质资源曲叶病毒检测分析初报

本研究依托"第三次全国农作物种质资源普查与收集行动",利用巢式PCR(Nested PCR)检测技术,对从湖南各地区采集的甘薯种质资源进行甘薯曲叶病毒的调查、检测、统计与分析,获得该地区甘薯种质资源曲叶病毒的感染和分布情况。对收集的246份甘薯种质资源进行了甘薯曲叶病毒病症状的调查,记录了每份种质资源的田间生长特性;建立了一种甘薯曲叶病毒巢式PCR检测技术,该技术相对其他技术具有特异性强、灵敏度高、检测通量大、检测成本低的特点。利用建立的巢式PCR检测技术对选取的样品进行甘薯曲叶病毒检测,分析检测结果发现:(1)巢氏PCR共检测出14份甘薯种质资源感染甘薯曲叶病毒,根据病毒基因测序结果分析湖南省至少存在2种曲叶病毒株系。(2)田间调查共发现8份甘薯种质资源的叶片具有甘薯曲叶病毒病典型的卷曲症状,但是其中仅有4份资源与曲叶病毒巢氏PCR检测结果一致;另外4份资源虽然具有明显的卷叶现象但是未检测出曲叶病毒。(3)曲叶病毒检测呈阳性的14份甘薯种质资源分别来源于邵阳市、长沙市、永州市和株洲市4个地区,占种质资源总数的5.7%;4个地区甘薯种质资源的病毒感染率分别为17.6%、14.5%、7.1%和6.7%;全省范围内的种质资源染病情况具有较大的地域差异性;综合甘薯种植情况和地理环境分析,商品薯的跨区域流通和农民自留种的种植习惯是影响甘薯病毒传播的主要因素。本研究首次利用巢式PCR技术对湖南地区甘薯曲叶病毒进行检测和调查,为甘薯种质资源的保存、繁殖、鉴定与利用提供了重要的技术支撑,也为湖南地区甘薯曲叶病毒侵染情况及相关的分子生物学研究提供了数据参考。     

甘薯叶柄原生质体有效植株再生

将甘薯（Ｉｐｏｍｏｅａｂａｔａｔａｓ（Ｌ．）Ｌａｍ．）‘元气’和‘白星’（‘ＷｈｉｔｅＳｔａｒ’）的叶柄原生质体培养在含有０．０５ｍｇ·Ｌ－１２,４Ｄ和０．５ｍｇ·Ｌ－１ＫＴ的改良ＭＳ液体培养基中,３～４ｄ后细胞开始分裂。培养８～９周后,将直径达１～２ｍｍ的愈伤组织转移到添加０．０５～０．２ｍｇ·Ｌ－１２,４Ｄ和０～０．５ｍｇ·Ｌ－１ＫＴ或添加０．５～２．０ｍｇ·Ｌ－１ＮＡＡ和１．０～３．０ｍｇ·Ｌ－１ＢＡＰ的ＭＳ固体增殖培养基上使其增殖。转移３～５周后,将愈伤组织再转移到ＭＳ基本培养基或转移到添加２．０～３．０ｍｇ·Ｌ－１ＢＡＰ的ＭＳ培养基上。当进一步转移到ＭＳ基本培养基上后,从愈伤组织或从愈伤组织形成的不定根上再生出植株。‘元气’植株再生率高达６０．０％,ＷｈｉｔｅＳｔａｒ高达４３．４％。     

甘薯生物技术研究进展

新兴的生物技术为甘薯这一古老的农作物带来了新的发展契机。细胞大规模培养、体细胞融合、基因转导等技术的研究和应用 ,可望从根本上改变甘薯传统的生产和育种模式。本文综合近年来国内外甘薯体细胞胚胎发生、原生质体培养和基因工程等方面的研究进展 ,对影响甘薯体胚发生体系及原生质体再生体系建立的诸多因素进行了详细论述 ,讨论了甘薯基因工程研究的应用潜力和目前存在的一些问题。     

Biosorption of Heavy Metals from Wastewater by Biosolids

In a study where the removal of heavy metals from wastewater is the primary aim, the biosorption of heavy metals onto biosolids prepared as Pseudomonas aeruginosa immobilized onto granular activated carbon was investigated in batch and column systems. In the batch system, adsorption equilibriums of heavy metals were reached between 20 and 50 min, and the optimal dosage of biosolids was 0.3 g/L. The biosorption efficiencies were 84, 80, 79, 59 and 42 % for Cr(VI), Ni(II), Cu(II), Zn(II) and Cd(II) ions, respectively. The rate constants of biosorption and pore diffusion of heavy metals were 0.013–0.089 min^–1 and 0.026–0.690 min^–0.5. In the column systems, the biosorption efficiencies for all heavy metals increased up to 81–100 %. The affinity of biosorption for various metal ions towards biosolids was decreased in the order: Cr = Ni > Cu > Zn > Cd.     

2，4_D对甘薯体细胞胚胎发生的调控

将来源于‘徐薯１８’叶片的胚性愈伤组织,接种在含有不同２,４Ｄ浓度的液体ＭＳ培养基中进行悬浮培养,悬浮细胞表现出不同的形态结构、分裂方式和发育途径：２,４Ｄ浓度为１ｍｇ／Ｌ时,细胞均等分裂,增殖迅速;不含２,４Ｄ时,细胞多进行不均等分裂,并发育成体细胞胚。不同２,４Ｄ浓度中培养的悬浮细胞,其胞外过氧化物同工酶谱及其随时间变化的方式有很大差异,并与细胞的生长、发育过程密切相关     

Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli

Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli.Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (μRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search. CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.     

Rapid and Efficient Purification of Haemolymph Ferritin from Cricket, Gryllus bimaculatus

The Gryllus bimaculatus ferritin was purified from the haemolymph by a consecutive four‐step procedures consisting of 50% ammonium sulfate fractionation, anion exchange column chromatography using HiTrap^TM Q column (1.6 x 4 cm, Amersham Bioscience), 70°C heat treatment for 10 min, acid treatment of 0.1 M sodium acetate buffer (pH 6.0), and gel filtration column chromatography using G4000SW column (0.75 x 60 cm, Tosoh, Japan) connected on FPLC system. The purified ferritin was found to have two major subunits of 32 and 30 kDa and three minor subunits of 28, 27, and 25 kDa by 2D electrophoresis analysis. Amino acid composition analysis showed that there are high contents of Asp, Glu, Met, Leu, and Lys residues in ferritin while low contents of Cys, Tyr, and Trp residues in the protein. G. bimaculatus haemolymph ferritin could be classified as a methionine‐rich protein.     

Removal of Heavy Metals from the Environment by Biosorption

The pollution of the environment with toxic metals is a result of many human activities, such as mining and metallurgy, and the effects of these metals on the ecosystems are of large economic and public‐healthsignificance. This paper presents the features and advantages of the unconventional removal method of heavy metals – biosorption – as a part of bioremediation. Bioremediation consists of a group of applications, which involvethe detoxification of hazardous substances instead of transferring them from one medium to another, by means of microbes and plants. This process is characterized as less disruptive and can be often carried out on site, eliminating the need to transport the toxic materials to treatment sites. The biosorption (sorption of metallic ions from solutions by live or dried biomass) offers an alternative to the remediation of industrial effluents as well as the recovery of metals contained in other media. Biosorbents are prepared from naturally abundant and/or waste biomass. Due to the high uptake capacity and very cost‐effective source of the raw material, biosorption is a progression towards a perspective method. The mechanism by which microorganisms take up metals is relatively unclear, but it has been demonstrated that both living and non‐living biomass may be utilized in biosorptive processes, as they often exhibit a marked tolerance towards metals and other adverse conditions. One of their major advantages is the treatment of large volumes of effluents with low concentrations of pollutants. Models developed were presented to determine both the number of adsorption sites required to bind each metal ion and the rate of adsorption, using a batch reactor mass balance and the Langmuir theory of adsorption to surfaces or continuous dynamic systems. Two main categories of bioreactors used in bioremediation – suspended growth and fixed film bioreactors – are discussed. Reactors with varying configurations to meet the different requirements for biosorption are analyzed considering two major groups of reactors – batch reactors and continuous reactors. Biosorption is treated as an emerging technology effective in removing even very low levels of heavy metal.     

甘薯丛枝病植原体的PCR检测

以报道的植原体（Phytoplasma)16SrDNA基因保守序列为依据,设计合成了两对引物对R16mF2/R16mR2和R16F2／R16R2,以甘薯丛枝病（SPWB）带病植株的叶脉中提取的DNA为模板,应用聚合酶链式反应（PCR）技术和巢式PCR（Nested-PCR)技术对甘薯丛枝病病原进行分子检测。结果表明PCR扩增出了1．5kb的特异片段,在PCB基础上的巢式PCR扩增出了1．2kb的特异片段,灵敏度实验显示该方法所需PCR模板DNA量为0．1073ng/ul在PCR的基础上的巢度PCR可以将灵敏度提高约10000倍,所需模板DNA仅为0．01073pg/ul,在甘薯丛枝病的检测中是一种快速,灵敏,可靠的方法。