野蚕黑卵蜂寄主识别利它素的纯化及氨基酸组成分析

针对利它素特殊的物理及化学性质 ,采用特定的温度分离方法 ,成功地从野蚕Theophilamandarina和家蚕Bombyxmori的雌蛾性附腺中分别获得了纯度较高的野蚕黑卵蜂Telenomustheophilae寄主识别利它素。对这两种利它素氨基酸组成的分析表明 ,来自家蚕和野蚕雌蛾性附腺的利它素在氨基酸的组成和含量方面非常相似 ,在检测到的 15种氨基酸中 ,甘氨酸、谷氨酸和天冬氨酸的克分子百分数在 10 %以上。从家蚕得到的分别为 2 8 1%、 18 5 %和 12 6 %。从野蚕得到的分别为 2 4 4%、18 1%和 10 1%。这 3种氨基酸之和在家蚕和野蚕中均超过 5 0 %以上。用凝胰乳蛋白酶对这两种利它素进行水解时发现溶液中均产生非水溶性沉淀 ,电泳表明沉淀的多肽蛋白分子量在 10kD左右。这显示在野蚕和家蚕利它素结构中 ,有着与家蚕丝蛋白相类似的结构 ,存在着结晶区域 (沉淀 )和无定形区域 (上清 )。     

家蚕性附腺发育与核酸及利它素蛋白变化的关系

对不同发育时期家蚕Bombyx mori雌性性附腺核酸和蛋白质含量测定的结果表明, 从家蚕化蛹后第6天到成虫羽化当天的性附腺内总蛋白质含量不断增加,至羽化当天为最高,达860±70 μg/对。不同时期内能引诱野蚕黑卵蜂识别寄主的利它素在总蛋白中所占的比例差异明显,从化蛹后第6天的10%增加到羽化后的58%。性附腺的总RNA含量从化蛹第6天到成虫羽化前1天几乎是直线增加,但羽化后下降很快。不同时期分泌部中总RNA含量变化与贮存部明显不同。含量最高时,分泌部RNA可占整个性附腺RNA的90%以上,而贮存部总RNA含量则远少于分泌部,羽化当天约为分泌部的十分之一。分泌部总RNA中的18S亚基含量远高于28S亚基,明显不同于贮存部的。从分泌部总RNA中分离的mRNA存在明显的条带分布,这预示着高丰度的mRNA的存在与总蛋白中高含量利它素的存在有着特殊对应关系。     

野蚕黑卵蜂寄主识别利它素的cDNA文库构建

根据家蚕性附腺中含有的利它素能诱导野蚕黑卵蜂寄生涂有该利它素的人造卵及能用家蚕卵繁育野蚕黑卵蜂的特性 ,用化蛹 9天的家蚕雌蛹性附腺分泌部的高丰度的mRNA构建了富含利它素基因的cDNA文库。构建成的未扩增文库的滴度为 3.2 9× 10 4 pfu μL ,重组率为 90 .0 5 % ,扩增后的文库总滴度为 1.5 6× 10 7pfu μL。用PCR方法对总文库和随机挑取的噬菌斑进行扩增 ,表明λZiplox载体中已含有大小为 0 .5～ 8.0kb的cDNA插入片段 ,并在 2 .5kb、1.1kb和 0 .75kb处cDNA插入片段相对集中。所有分析结果表明 ,采用特定的发育时期和特定的组织提取的mRNA构建而成的cDNA表达文库具有较高频率的利它素基因片段和合适的滴度 ,定向插入技术并结合λZiplox噬菌体的表达功能 ,使构建的cDNA文库具有表达外源DNA的功能 ,有利于进行利它素基因的免疫筛选。     

家蚕雌性附腺及其Ng突变体的蛋白质组差异研究

家蚕雌蛾性附腺在化蛾前2到3天开始大量分泌胶状粘性蛋白,其贮存部迅速地膨大,而其Ng突变体的雌蛾性附腺不能正常分泌胶状粘性物质.分别对家蚕(Bombyx mori)的正常及Ng突变体雌蛾性附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析,并对主要差异表达的蛋白质用质谱鉴定.实验结果表明,用银染法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH 4～8范围内,其分子质量主要集中在30～70 ku区域.比较分析发现一些差异表达蛋白,其中No2,3蛋白质点经质谱鉴定为肌动蛋白A3,该蛋白质只在化蛹后期正常雌性附腺组织中特异表达,而Ng突变体中肌动蛋白A3的缺失,暗示了肌动蛋白A3可能与家蚕雌性附腺的胶状粘性物质的胞外分泌有关.     

家蚕雌性附腺及其Ng突变体的蛋白质组差异研究(英)

家蚕雌蛾性附腺在化蛾前2到3天开始大量分泌胶状粘性蛋白,其贮存部迅速地膨大,而其Ng突变体的雌蛾性附腺不能正常分泌胶状粘性物质.分别对家蚕（Bombyx mori）的正常及Ng突变体雌蛾性附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析,并对主要差异表达的蛋白质用质谱鉴定.实验结果表明,用银染法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH 4～8 范围内,其分子质量主要集中在30～70 ku区域.比较分析发现一些差异表达蛋白,其中No2, 3蛋白质点经质谱鉴定为肌动蛋白A3,该蛋白质只在化蛹后期正常雌性附腺组织中特异表达,而Ng突变体中肌动蛋白A3的缺失,暗示了肌动蛋白A3可能与家蚕雌性附腺的胶状粘性物质的胞外分泌有关.     

中国野蚕一种强抗病毒蛋白的基因分析和活性鉴定

以最近报道的家蚕抗病毒蛋白基因为线索,从中国野蚕（Bombyx mandarina Moore）中肠内克隆了抗家蚕BmNPV病毒的SP-2 cDNA（GenBank登录号：AY945210）,基因大小855bp,编码284个氨基酸的蛋白质,分子量29．6kD,基因组全长1376bp,包含5个外显子和4个内含子．该基因的表达仅限于中肠,具有组织特异性,在幼虫龄中表达水平较高,而在眠期和熟蚕没有表达．推导其氨基酸序列,发现其C端氨基酸序列与已报道的家蚕相应序列差别较大,有8个氨基酸完全不同．通过体外重组技术,由高效基因表达系统获得大量重组蛋白,发现该蛋白具有很强的抗家蚕BmNPV活性,与家蚕对应的抗病毒蛋白BmSP-2相比,其抗BmNPV活性高1．6倍．初步认为,该蛋白质C端序列差异可能是造成家蚕与野蚕抗病毒活性差别的主要原因．     

挥发性信息化合物对玉米螟赤眼蜂寄主选择行为的影响

在实验室条件下,利用“Y”型嗅觉仪测定了源于寄主亚洲玉米螟Ostrinia furnacalis (Guenée) 鳞片、卵表以及不同生理阶段的雌蛾附腺的不同浓度的提取物对玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen寄主选择行为的影响,并对其引诱作用大小进行了比较。结果表明： 亚洲玉米螟鳞片的正己烷提取物在0.5 mg/mL和 1 mg/mL浓度时对玉米螟赤眼蜂有明显的引诱作用; 寄主卵的正己烷提取物在5块卵/mL和10块卵/mL浓度时对玉米螟赤眼蜂有显著的引诱作用,而在40块卵/mL浓度时对玉米螟赤眼蜂有极显著的驱避作用; 玉米螟赤眼蜂对亚洲玉米螟交配未产卵和产卵后前期的雌蛾附腺提取液有反应,而对处女蛾和产卵后期的附腺提取液没反应。卵表提取物和附腺提取物比鳞片提取液对赤眼蜂引诱作用强,两者对赤眼蜂的引诱作用没有明显差异。     

玉米螟赤眼蜂对亚洲玉米螟益它素的嗅觉反应

在实验室条件下利用四臂嗅觉仪测定了玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen对来源于寄主亚洲玉米螟Ostrinia furnacalis (Guenée)成虫、卵、附腺以及性信息素的益它素的嗅觉反应。结果表明: 已交配尚未产卵亚洲玉米螟雌蛾及其附腺对玉米螟赤眼蜂有明显的吸引作用,而处女蛾、产卵前期雌蛾、产卵后期雌蛾及其附腺却没有作用;当玉米螟卵块数量为2块及卵表正己烷提取物当量为0.25块卵时,对玉米螟赤眼蜂也有明显的吸引作用,尤其以产在玻璃管内卵块吸引最为显著,达到极显著水平(P<0.01);性信息素组分之一,反-12-十四碳烯乙酸酯(E12.14: Ac)在剂量为1 μg时亦显著刺激了赤眼蜂的活动,表现为进入处理区的次数明显增加,且滞留时间明显延长。     

家蚕雌性附腺及其Ng突变体蛋白质组双向电泳图谱分析

分别对家蚕(Bombyx mori．L)正常及Ng突变体雌蛾件附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析。用银染的方法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH4～8范围内,在分子量上主要集中在30～70kD区域：比较分析发现,有4种蛋白只在正常性附腺组织中特异表达,而有2种蛋白只在Ng突变体的组织中特异表达。另外约有29种蛋白在正常性附腺分泌部组织中的表达水平明显高于Ng突变,而约有15种蛋白在Ng突变体的分泌部组织中表达水平较高。这些差异蛋白质可能与Ng突变的形成和导致这种突变体的性附腺不能正常分泌粘性蛋白的性状有关。     

带有丝素重链信号肽序列的家蚕丝胶蛋白启动子驱动DsRed的瞬时分泌表达

为了探讨家蚕Bombyx mori丝素蛋白重链信号肽序列在中部丝腺组织中是否具有功能活性,根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,构建了带有丝素重链基因fib-H信号肽的家蚕丝胶-1(ser-1)启动子(ser-HS),用ser-HS驱动DsRed基因构建了分泌型瞬时表达载体pSK-SerHS-DsRed-polyA。转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed。家蚕注射载体后,可在中部丝腺腔中检测到红色荧光,表明瞬时表达的DsRed已分泌到丝腺腔内。据此提出克隆的fib-H信号肽序列在家蚕中部丝腺组织中具有信号肽的功能。     

Evaluation of methods for the separation and analysis of proteins and free amino acids in phytoplankton samples

Due to their importance as not only major constituents in paniculatematter but also the metabolism of nitrogen in marine microorganisms,numerous methods have been employed to measure proteins andfree amino acids. However, two difficulties frequently complicatethese measurements. First, an initial separation of proteinsfrom free amino acids is helpful since most analytical methodsare somewhat sensitive to both compound types. Second, the choiceof detection techniques that minimize response differences betweenvarious proteins or amino acids is desirable since natural samplesof microorganisms consist of mixtures of many proteins and aminoacids. To address these problems, four protein detection techniques(modified Lowry et al., Dorsey et al., Bradford and fluorescamine)and two amino acid detection techniques (fluorescamine and o-phthaldialdehyde)were evaluated. Relative extraction efficiencies for proteinfrom phytoplankton samples were also evaluated with six homogenizationsolutions/protocols (TCA, NaOH, boiling NaOH, Triton X-100,NaOH plus Triton X-100 and distilled water). TCA homogenizationyielded the highest protein recoveries, and sufficient physicalseparations between proteins and free amino acids were obtainedwith TCA concentrations between 0.18 and 0.37 M. Results ofthese studies allowed for development of a method for extracting,separating and analyzing proteins and total free amino acidsfrom a common phytoplankton sample. The procedure involves initialhomogenization in a TCA solution, followed by centrifugationto separate protein and free amino acid fractions. Proteinsare then analyzed by a modification of the Lowry et al. procedure,and amino acids by a fluorescamine procedure. ²Present address: Science Applications International Corporation,4224Campus Point Court, San Diego, CA 92121, USA     

Polypeptide Compositions and NH2-terminal Amino Acid Sequences of Proteins in Foxtail and Proso Millets

Seed protein of foxtail and proso millets were fractionated into polypeptides that were analyzed for their major protein, prolamin, and the NH₂-terminal amino acid sequences of the proteins were determined. The proteins extracted from foxtail and proso millets were 64.1% and 80.0% prolamin, respectively. The polypeptides of the prolamins were classified into two groups. The major polypeptides of 27-19 kDa were rich in leucine and alanine, whereas the 17-14 kDa polypeptides were rich in methionine and cysteine. Glutelin-like proteins that were extracted with a reducing reagent were high in proline content, the major polypeptides being 17 and 20 kDa. The NH₂-terminal amino acid sequence showed that the major polypeptides of prolamin were homologous to α-zein and a glutelin-like protein containing the Pro-Pro-Pro sequence, like the repetitive sequence of γ-zein. Although the prolamin consisted of a similar subunit to that of zein, polypeptides with various pI values were found among them.     

Control of Seed Protein Accumulation in Field Bean (Vicia faba L.)

A study was made of the changes during development in the totalamino acid and 3, 4 dihydroxyphenylalanine (DOPA) content ofbean pod phloem sap, employing EDTA to aid phloem exudation.Two field bean lines, Dacre B and D, selected for their lowand high seed protein content respectively, were compared. Throughoutdevelopment, the sap samples of Dacre D had a greater aminoacid concentration than those from Dacre B. The sap of DacreB contained a higher proportion of DOPA than that of Dacre D.These two lines of Dacre were also studied with respect to accumulationof protein and uncombined amino acid in cotyledons grown bothin vitro and in vivo. Dacre D accumulated more total proteinthan Dacre B but contained a similar amount of uncombined aminoacids when grown in vivo. However, the amount of total proteinaccumulated was similar when the cotyledons were grown in vitro.The data suggest that the supply of nutrients to the pod maybe the basis of the different protein concentrations in themature seed of these lines. Vicia faba L., field bean, phloem sap, cotyledon culture, amino acids, DOPA, protein     

cDNA cloning and mRNA expression of LIM protein gene homologue from the silkworm, Bombyx mori

LIM protein cDNA, from Bombyx mori that contains an open reading frame of 622 bp encoding 94 amino acids, was identified and characterized. The B. mori LIM protein homologue is classified into group 2 LIM proteins that contain glycine-rich LIM domain. B. mori LIM protein mRNA is up-regulated at late embryogenesis and detected in the mid-gut of 5th instar larvae.     

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.     

Effects of Nutrient Supply on Seed Growth of Field Bean (Vicia faba L.). 1. Effects of Different Sulphur and Methionine Regimes on Cotyledon Growth, Protein and Uncombined Amino Acids In vitro

The effects of differemt S and methionine regimes on growthof developing Vicia faba cotyledons in vitro were studied. Basalmedium (containing adequate S) supplemented with 05 mM methioninemarginally increased d. wt and uncombined amino acid accumulationbut adding 1–5 mM methionine inhibited both growth andprotein accumulation. Sulphur deficiency reduced both d. wtand protein accumulation but incresed accumulation of uncombinedamino acids. Adding 1 mM methionine to the S-deficient mediumrestored growth, normal protein and uncombined amino acid acnunulation.High sulphate medium (7.5 mM ) decreased d. wt, protein anduncombined amino acid accumulation. High sulphate medium or basal medium+methionine (05 mM) changedthe proportions of the seed proteins; legumin increased butvicilin decreased. Sulphur deficiency caused a relative increasein vicilin but a decrease in legumin. The different S and methionineregimes markedly changed the composition of the uncombined aminoacids, especially those derived from aspartic acid but not thecomposition of the protein fraction, except during S deficiency. The data presented indicates a flexibility in the storage proteincomposition of developing cotyledons grown in vitro, with theS and methionine status having a regulatory effect. Vicia faba L., field bean, cotyledon, growth, in vitro culture, uncombined amino acids, protein composition, legumin, vicilin, methionine, sulphur     

甜菜夜蛾泛素延伸蛋白基因cDNA的3′RACE-PCR扩增及其克隆和表达

根据已知的草地夜蛾Spodoptera frugiperda的泛素延伸基因 5'端核苷酸序列设计引物,应用3'RACE-PCR技术,从甜菜夜蛾S. exigua脂肪体组织总RNA中反转录扩增泛素基因的cDNA片段。扩增得到的片段全长513 bp,3'末端有123 bp的非翻译区,翻译区编码一个长为129个氨基酸残基的蛋白质,预测分子量为14.8 kD。同源分析表明,此cDNA序列为ubiquitin-53aa extension protein(ubi-53) 基因,在泛素蛋白后融合了一个核糖体L40蛋白（ribosomal L40 protein）。用MagAlign和Genedoc软件对cDNA编码的氨基酸序列进行了同源性分析,结果表明： 甜菜夜蛾的ubi-53基因与真核生物家蚕Bombyx mori、草地夜蛾、果蝇Drosophila melanogaster和人Homo sapienes泛素的同源性分别为96.9%、98.5%、95.3%和93.0%,与甜菜夜蛾核型多角体病毒(SeNPV)泛素的同源性为78.8%,说明真核生物的泛素基因与核型多角体病毒的泛素基因可能存在不同的分子进化途径。将甜菜夜蛾的ubI-53基因克隆到原核表达载体pET-28a上,转化至BL21（DE3）中,用IPTG进行诱导表达,用异源泛素单克隆抗体进行Western blot检测,证明原核表达蛋白是目的蛋白。     

Effect of Abscisic Acid, Osmoticum, and Desiccation on Synthesis of Storage Proteins during the Development of White Spruce Somatic Embryos

The effect of abscisic acid (ABA), non-permeating osmoticumand desiccation treatment on storage protein synthesis duringmaturation of somatic embryos of Picea glauca (Moench) Voss.was examined. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis demonstrated that someof the major crystalloid and matrix polypeptides were absentfrom somatic embryos maturing on medium containing ABA and lowosmoticum. However, treatment with polyethylene glycol-4000(PEG) in combination with ABA resulted in the synthesis of aspectrum of storage polypeptides resembling that of mature zygoticembryos. These storage proteins accumulated throughout an 8-weekculture period, resulting in a threefold higher protein contentthan somatic embryos maturing for the same time in the absenceof PEG. The structure and distribution of protein bodies incells of these osmotically treated somatic embryos was similarto that in cells of mature zygotic embryos. Treatment with 5·0-7·5%PEG prevented catabolism of the accumulated storage polypeptidesduring desiccation. The optimal culture conditions for somaticembryo maturation and storage protein deposition was 16 µMABA and 7·5% PEG for 8 weeks followed by desiccation.Analysis of mRNAs by in vitro translation and immunoprecipitationof translated products showed that the crystalloid protein mRNAprofiles of zygotic and those of somatic embryos maturing on16 µM ABA in the absence of PEG were similar. The differencesobserved in the pattern of accumulated polypeptides in thesesomatic embryos and those of mature zygotic embryos, therefore,indicates that storage-protein synthesis in response to osmoticumis in part regulated at the translational level. During regenerationof somatic embryos to plantlets the storage polypeptides wererapidly utilized in a manner similar to that in zygotic seedlings.Copyright1993, 1999 Academic Press Desiccation, osmotic stress, storage proteins, Picea, embryogenesis—somatic, mRNA (crystalloid protein)     

Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L

Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M_r. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds.