不同品种花生种子蛋白质的电泳分析

对中国花生（ＡｒａｃｈｉｓｈｙｐｏｇａｅａＬ．）５种不同类型的４６个栽培品种的种子蛋白质进行电泳分析。ＳＤＳＰＡＧＥ和ＩＥＦＳＤＳＰＡＧＥ（双向电泳）的结果显示,不同品种的花生种子蛋白多肽组成存在４种类型。与初步纯化的花生球蛋白及伴花生球蛋白的ＳＤＳＰＡＧＥ结果比较,花生种子蛋白组成的主要差异在于花生球蛋白亚基组成的不同,其中类型Ⅰ花生球蛋白主要含有４１ｋＤ、３８５ｋＤ和２个１８ｋＤ亚基,类型Ⅱ主要含４１ｋＤ、３８５ｋＤ、３７５ｋＤ和３个１８ｋＤ亚基,类型Ⅲ主要含４１ｋＤ、３８．５ｋＤ、３６．５ｋＤ和３个１８ｋＤ亚基,类型Ⅳ主要含４１ｋＤ、３８．５ｋＤ、３７５ｋＤ、３６．５ｋＤ和３个１８ｋＤ亚基。不同品种的伴花生球蛋白多肽组成基本一致。对不同类型８个品种种子蛋白的氨基酸组成进行测定,发现类型Ⅰ的含硫氨基酸含量较高。     

不同品质类型花生品质性状及相关酶活性差异

在大田栽培条件下,以高蛋白花生品种KB008、高脂肪品种花17(H17)和高油酸/亚油酸(O/L)品种农大818(818)为试验材料,研究了3种类型花生品种籽仁中蛋白质、脂肪含量及与品质合成相关的碳、氮代谢酶活性差异.结果表明:KB008的蛋白质含量显著高于H17和818,而可溶性糖含量和O/L值显著低于其他两品种.KB008籽仁中氨基酸组分含量均高于其他两品种,特别是谷氨酸和赖氨酸含量显著高于后两者;油酸含量显著低于、而亚油酸含量显著高于其他两品种.3种类型花生在整个生育期中叶片的硝酸还原酶(NR)、谷氨酰胺合成酶(GS)、谷氨酸脱氢酶(GDH)、谷氨酸合成酶(GOGAT)和谷丙转氨酶(GPT)活性均以KB008最高,其次为H17.3种类型花生在结荚期的叶片磷酸烯醇式丙酮酸羧化酶(PEPCase)和1,5-二磷酸核酮糖羧化酶(RuBPCase)活性大小均表现为KB008 ＞H17 ＞818,说明较高的PEPCase和RuBPCase活性有利于蛋白质合成与积累.叶片中蔗糖合成酶(SS)活性大小表现为H17＞818＞KB008,KB008的磷酸蔗糖合成酶(SPS)活性显著低于其他两品种,而H17的SPS活性在花后60d时仍保持较高活性,说明较高的叶片SPS、SS活性有利于花生籽仁脂肪的形成.     

红豆草抗甲硫氨酸变异体的筛选

用ＮａＮ３ 诱变过的红豆草（Ｏｎｏｂｒｙｃｈｉｓｖｉｃｉａｅｆｏｌｉａ Ｓｃｏｐ．）愈伤组织筛选得到了能抗８０ ｍ ｍ ｏｌ／Ｌ甲硫氨酸的变异细胞系——ＯＮｍ ｅｔｒ,并得到了再生植株。ＯＮｍ ｅｔｒ 脱离选择压力６ 个月后,其抗性仍比野生型的高５．６ 倍。同时还表现出对乙硫氨酸的交叉抗性,其抗性是野生型的６．５ 倍, 表明该抗性系抗性表达稳定。ＯＮｍ ｅｔｒ 愈伤组织中甲硫氨酸、赖氨酸、苏氨酸含量分别是野生型的４．００、１．０９、１．５０ 倍。ＯＮｍ ｅｔｒ再生植株中甲硫氨酸、赖氨酸、苏氨酸、异亮氨酸含量分别为野生型的２．０、３．５、３．５、２．５ 倍。在ＯＮｍ ｅｔｒ 愈伤组织可溶性蛋白ＳＤＳ－ＰＡＧＥ图谱中,出现了两条新多肽（３０ ｋＤ、２６ ｋＤ）。ＯＮｍ ｅｔｒ 细胞系过氧化物酶同工酶谱中亦出现了两条新酶带。这些变化表明该变异系已经产生变化了的基因产物     

丝孢酵母高甲硫氨酸突变株的选育及营养调控

以丝孢酵母(Trichosporon Behr)ST851为原始菌株,经紫外线诱变,在含乙硫氨酸的双层平板上筛选到多株抗乙硫氨酸突变株。其中ST851-10株抗乙硫氨酸浓度达到350μg/ml,其菌体蛋白质含量由40.5％提高到44.3％,菌体甲硫氨酸含量由20.45mg/g-DCW增加到29.32mg/g-DCW。在以苹果渣为碳源、尿素为氮源、硫酸镁作硫源的最适培养条件下,固态发酵24h后,蛋白质和甲硫氨酸含量较原始菌株分别提高了15.8％和44.9％。培养基中C/N值低有利于甲硫氨酸的合成,C/N值高则适合于菌体生长。在苹果渣固态发酵过程中,适当补加氮源既有利于菌体生长和甲硫氨酸的合成,又可起到调节培养基pH值的作用。     

海南岛绞股蓝属植物中总氨基酸分析

应用日立８３５５０型氨基酸自动分析仪对海南岛不同生境绞股蓝属５种植物中的总氨基酸组分进行了含量测定与比值分析。结果表明：绞股蓝（５４．１１％）＞单叶绞股蓝（５３．８０％）＞光叶绞股蓝（４４．９８％）＞喙果绞股蓝（３５．５８％）＞毛果绞股蓝（１７．５０％）。各种样品均含有１７种氨基酸,Ｇｌｕ、Ｓｅｒ、Ｇｌｙ和Ａｓｐ含量最高,Ｍｅｔ、Ｃｙｓ、Ｈｉｓ偏低;栽培的高于野生的,量值相比变化范围达０．７％～２．７０９％;５种绞股蓝游离氨基酸定量分析结果,以单叶绞股蓝和绞股蓝含量最高（１３．４０５％、１２．９０４％）、喙果绞股蓝与光叶绞股蓝居中（１１．４８％、１１．１６３％）、毛果绞股蓝稍为偏低（７．６８０％）。     

海南岛绞股蓝植物中总氨基酸分析

应用日立８３－５０型氨基酸自动分析仪对海南岛不同生境绞股蓝属５种植物中的总氨基酸组分进行了含量测定比值分析。结果表明：绞股蓝（５４．１１％）〉单叶绞股蓝（５３．８０％）〉光叶绞股蓝（４４．９８％）〉果绞股蓝（３５．５８％）〉毛果绞股蓝（１７．５０％）。各种样品均含有１７种氨基酸,Ｇｌｕ、Ｓｅｒ、Ｇｌｙ和Ａｓｐ含量最高,Ｍｅｔ、Ｃｙｓ、Ｈｉｓ偏低;栽培的高于野生的。量值相比变化范围达０．７－２．７０     

多效唑对不同品质类型花生产量、品质及相关酶活性的影响

选用高蛋白品种KB008（KB008）、高脂肪品种花17(H17)和高油酸/亚油酸（O/L）品种农大818（818）,在大田栽培条件下,研究了盛花后期叶面喷施多效唑(PBZ)对不同品质类型花生产量、品质及相关碳、氮代谢酶活性的影响.结果表明：喷施PBZ显著增加了3种品质类型花生荚果产量,原因是增加了单株结果数,降低了千克果数而提高了双仁果率.喷施PBZ不同程度地提高了3种类型花生籽仁脂肪和可溶性糖含量,降低了蛋白质含量,显著增加了高脂肪品种H17的O/L值.PBZ使高O/L值品种818的脂肪含量增加显著,同时其蛋白质含量显著降低,而对其他两品种的蛋白质和脂肪含量影响较小.喷施PBZ均降低了3种类型花生结荚期叶片硝酸还原酶（NR）活性及结荚期和饱果期叶片谷氨酰胺合成酶和谷氨酸脱氢酶活性,818的3种酶活性降低幅度最大,KB008和H17的酶活性降幅较小;喷施PBZ均降低了3种类型花生结荚期和饱果期叶片谷草转氨酶和谷丙转氨酶活性.说明氮代谢酶活性的降低是喷施PBZ降低3种类型花生籽仁蛋白质含量的主要原因.喷施PBZ均提高了3品种结荚期和饱果期叶片蔗糖合成酶和磷酸蔗糖合成酶活性,其中显著提高了818的2种酶活性,而对KB008和H17的活性提高不显著;喷施PBZ提高了3品种结荚期和饱果期的磷酸烯醇式丙酮酸羧化酶和1,5-二磷酸核酮糖羧化酶活性,其中对818在结荚期的活性提高最显著,对H17活性提高较小.碳代谢酶活性的增强是喷施PBZ提高花生籽仁脂肪含量的生理基础.     

唐松草幼苗氨基酸,无机元素的测定

唐松草氨基酸含量的分析表明,１６种氨基酸总量为１８２．２ｇ／ｋｇ,尤其谷氨酸和天门冬氨酸含量较高,适口性好。必需氨基酸占总氨基酸的３４．２６％。无机元素测定结果表明唐松草１７种无机元素中磷含量最高达８７２．０μｇ／ｇ,其次是钙镁元素,且含有锌、锰、锶、铁等多种微量元素。同时也测定了蛋白质和粗脂肪的含量     

不同品种柑橘果肉中氨基酸的测定与分析

采用日立L-8800型氨基酸自动分析仪,测定5个品种柑橘果肉氨基酸含量。结果表明,柑橘果肉中富含天门冬氨酸、脯氨酸、谷氨酸。脐橙52果肉中总氨基酸含量(7.04 g·100g-1)较高,红肉蜜柚最低(4.53 g·100g-1),前者是后者的1.5倍;从氨基酸组分来看,5个品种中,17种氨基酸含量最高的是天门冬氨酸(Asp);各品种氨基酸总含量(TAA)与必需氨基酸(EAA)含量的波动趋势基本一致,即TAA含量越高,相应的EAA含量也高;品种间各类味觉类氨基酸含量的高低顺序并不完全一致,说明柑橘不同品种在营养学上有较大差异。     

黑曲霉纤维素酶的化学组成

本文测定的黑曲霉纤维素酶各组分均为糖蛋白,但含糖量和组成各不相同,含糖量分别为：β－葡萄糖苷酶１１．３％,内切β－葡聚糖酶１１．８％,β－葡聚糖纤维二糖水解酶ＣＢＨ—１７．２％、ＣＢＨ－Ⅱ５．８％。各酶组分的氨基酸组成有一定的差异,但也有一些共同之处,即都含有较多的酸性氨基酸（Ａｓｐ、Ｇｌｕ）,碱性氨基酸（Ｈｉｓ、Ａｒｇ）含量较低。分析酶组分间的相似性时发现,β－葡聚精纤维二糖水解酶的ＣＢＨ－Ⅰ组分与内切β－葡聚糖酶在各种氨基酸的含量上较为相似,而同属于β－葡聚糖纤维二糖水解酶的两个组分ＣＢＨⅠ和ＣＢＨ—Ⅱ在氨基酸的含量上有一定的差异。     

Suppression of phaseolin and lectin in seeds of common bean, Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

Suppression of phaseolin and lectin in seeds of common bean,Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

Comparison of the primary structure of the acidic polypeptides of glycinin

Some essential features of the primary structures of five acidic polypeptides from the major 11 S soybean storage protein were studied. Each purified polypeptide was cleaved at methionine using cyanogen bromide, and then each resulting fragment was purified. A comparison of the NH₂-terminal amino acid sequences of each fragment, coupled with an identification of both the carboxyl and amino terminal fragments, permitted ordering of the fragments along the polypeptides. It was found that the five acidic polypeptides were synthesized at the direction of a family of homologous genes. Evidence was also obtained which suggested that there were repeated domains of amino acid sequence within each of the five molecules.     

Synthesis and organization of vitellogenin and vitellin molecules from the land crab Potamon potamios

We have previously reported that vitellogenin (Vg) of some female animals contained four polypeptides with molecular mass of 181, 115, 105 and 85 kDa, whereas Vg of most animals contained three polypeptides with molecular mass of 115, 105 and 85 kDa. In the present investigation, we examined whether the 181 kDa polypeptide is the precursor of 115 and 105 kDa Vg and vitellin (Vn) polypeptides. Labeling studies, using [35S]methionine on normal vitellogenic animals, showed that the radioactivity was distributed first among the 181 and 85 kDa polypeptides. SDS-PAGE analysis of purified hemolymph Vg from eyestalk ablated female animals revealed in most animals two polypeptides with an apparent molecular mass of 181 and 85 kDa. These results from in vivo experiments corroborated the view that the 115 and 105 kDa Vg and Vn polypeptides are derived from heaviest 181 kDa polypeptide. In addition it was demonstrated that hepatopancreas and ovary of Potamon potamios incubated in vitro with [35S]methionine synthesized five polypeptides with apparent molecular mass of 224, 181, 115, 105, and 85 kDa while the hepatopancreas appeared to secrete the 181, 115, 105 and 85 kDa polypeptides. The major 115, 105 and 85 kDa polypeptides were found to be components of egg Vn, while the 224 kDa polypeptide was found to be minor component of Vg and Vn from hepatopancreas and ovary extracts, respectively. We conclude that the Vn polypeptides produced by ovary are similar to those produced by hepatopancreas.     

Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota.

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.     

One and Two- dimensional Polyacrylamide Gel Electrophoretic Analysis of Seed Polypeptide Composition of Peanut Cultivars

Seed polypeptides from 46 cultivars of peanut (Arachis hypogaea L. ) were compared by SDS-PAGE and two-dimensional pelyacrylamide gel electrophoresis. Arachin, the major seed storage protein of peanut, showed polymorphism. There were four types of arachin pelypeptide pattems. Type Ⅰ consisted of only four major subunits:41 kD,38.5 kD and two of the 18 kD subunits. Type Ⅱ had six major subunits:41 kD,38.5 kD,37.5 kD and three of the 18 kD subunits. Type Ⅲ consisted of 41 kD, 38.5 kD,36.5 kD and three of the 18 kD subunits. And Type Ⅳ consisted of seven major subunits:41 kD,38.5 kD,37.5 kD,36.5 kD and three of the 18 kD subunits. The compositions of conarachin in different cultivars were similar. Amino acid composition analysis of seed protein in 8 peanut cuhivars showed that Type Ⅰ was rich in methionine and cystine.     

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.     

5S-rRNA-containing ribonucleoproteins from rabbit muscle and liver. Complex and partial primary structures

A 5S-rRNA-containing ribonucleoprotein was purified to homogeneity from a rabbit muscle extract through its affinity to phosphofructokinase-1 and then structurally characterized. This RNP was compared to the 5S-rRNA-containing ribonucleoprotein extracted from rabbit liver ribosomal 60S subunits with EDTA. Analytical gel filtration revealed a molecular mass of 70-80 kDa for both complexes. Gel electrophoresis of the ribosomal complex revealed three protein components, one migrating as a band of 35 kDa and two other small polypeptides of apparently 16.5 kDa and 17.5 kDa. In the sarcoplasmic RNP these small polypeptides were absent. However, besides a major component of 35 kDa, up to five slightly larger and smaller species of 31.5-36.5 kDa were detected. Despite this heterogeneity, only one N-terminal amino acid sequence was obtained for the isolated sarcoplasmic protein, suggesting a C-terminal heterogeneity of one single polypeptide. Within the first 46 amino acid residues no difference between the sequences of the isolated 35-kDa components of sarcoplasmic and ribosomal complexes was found. Homology criteria indicated that this component belongs to the ribosomal protein L5 family. The RNA was identified by complete enzymatic sequencing as 5S rRNA; it was also identical in both complexes and is strongly homologous to 5S rRNA of man. Both L5-5S-RNA complexes could be resolved by hydroxyapatite chromatography into three species still consisting of both protein and RNA. 5'-Terminal dephosphorylation experiments showed that this heterogeneity is exclusively due to the differing number (1-3) of 5'-terminal phosphates. The two additional low-molecular-mass proteins were stably associated to the ribosomal RNP at high salt concentrations in a stoichiometry of about 2:1. They were identified as the acidic phosphoproteins P2/P3 by N-terminal sequencing. High phosphate concentrations facilitated their dissociation from the L5-5S-RNA complex. For the sarcoplasmic L5-5S-RNA complex a hitherto unknown interaction with phosphofructokinase-1, affecting the enzymatic properties, was demonstrated.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Molecular cloning and characterization of a cysteine-rich 16.6-kDa prolamin in rice seeds

An alcohol-soluble storage protein, a 16.6-kDa prolamin found in rice seeds, was purified from both the total protein body and purified type I protein body fractions. The partial amino acid sequences of three tryptic peptides generated from the purified polypeptide were analyzed. A part of the 16.6-kDa prolamin cDNA was amplified from developing seed mRNA by the reverse transcribed polymerase chain reaction using an oligo (dT) primer and a primer which was synthesized based on the partial amino acid sequence. The amplified product was used to isolate the full-length cDNA clone (lambda RP16) from a developing seed cDNA library. The cDNA has an open reading frame encoding a hydrophobic polypeptide of 149 amino acids. The polypeptide was rich in glutamine (20.0%), cysteine (10.0%), and methionine (6.9%). The cysteine content was higher than those of most other rice storage proteins. Messenger RNA of the 16.6-kDa prolamin was detected in seeds, but not in other aerial tissues.