绿叶蛋白的研制及开发利用前景

绿叶蛋白的研制及开发利用前景彭高禧（福建省闽东工业学校实验室宁德市３５２１００）生产一份食用蛋白（蛋类、奶类、肉类）要消耗５～１０倍甚至更多的饲用蛋白，在畜牧业和养禽业飞速发展的今天，饲用蛋白已呈全球性不足。据国外专家预测，到２０００年这个亏空的总数...

豆科绿叶蛋白不同原料、方法提取效果的研究

采用不同浓度梯度的乙醇、丙酮、硫酸铵和不同组合处理的酸化、碱化方法，分别从苜蓿、三叶草、小冠花和洋槐等豆科植物叶中提取叶蛋白。结果表明，苜蓿、三叶草、洋槐叶是制取叶蛋白的理想材料，三者的最高提取率分别为１２．５０ｇ／１００ｇ鲜草、１３．５０ｇ／１００ｇ鲜草和１５．６０ｇ／１００ｇ鲜草，提取方法以３０％硫酸铵处理最佳，７５％乙醇和酸化加热次之。同时，还提出了提取叶蛋白不同植物应该采用的处理方法。

黄豆叶蛋白营养价值的实验评价

目的：对黄豆叶蛋白质营养价值予以实验评价。方法：从黄豆的鲜叶中提取黄豆叶蛋白，测定粗蛋白、必需和半必需氨基酸，采用Ｗｉｓｔａｒ大鼠，喂饲黄豆叶蛋白配合饲料，以酪蛋白作为参考蛋白，以分离大豆蛋白作为对比物，参照ＡＯＡＣ方法，分析比较真消化率、生物价、蛋白质净利用率等指标。结果：黄豆叶粗蛋白含量为６１％，氨基酸评分（ＡＡＳ）为９８。黄豆叶蛋白的营养评价指标与分离大豆蛋白无差异，提示两者营养价值相似。结论：利用黄豆叶资源提取黄豆叶蛋白，是蛋白质食物来源的重要途径之一。

黄豆、豌豆叶蛋白的营养评价

采用纯系Ｗｉｓｔａｒ雄性大鼠，喂饲黄豆叶蛋白和豌豆叶蛋白配合的饲料，以酪蛋白作为参考蛋白，以分离大豆蛋白作为比照物，参照ＡＯＡＣ的方法，分析比较了ＴＤ、ＢＶ、ＮＰｕ、ＮＢ、ＰＥＲ和ＮＰＲ等指标。实验结果认为黄豆叶蛋白和豌豆叶蛋白的营养价值与分离大豆蛋白相似，是值得研究开发的蛋白质资源。

Effect of Foliar Fertilizer Spraying at Seedling Stage on the Growth Physiological Characteristics and Yield of Brassica napus L.

In order to select a suitable foliar fertilizer for Brassica napus L.at the seedling stage,using‘Fengyou 958’as the material,different foliar fertilizers including BR,Se,Si,BR+Si,BR+Se,Se+Si and BR+Se+Si were sprayed at the seedling stage to study their effect on the physiological characteristics,growth and yield of Brassica napus L..The results showed that the growth,chlorophyll content,soluble sugar content,soluble protein content,and yield of different treatments at the budding stage improved compared with the control.The effect of the Si+Se treatment was the best,followed by the Se and Si treatments.The chlorophyll content of the flower decreased continuously during the whole flowering period,and the chlorophyll content of the mature silique peel was higher than that of the seed.The content of soluble sugar in flowers was the highest in the early flowering stage,and the content of soluble sugar in leaves at the flowering stage was higher than that at the budding stage.The soluble sugar content in the harvested silique peel decreased gradually with the increase of time,and that in the silique peel and seed was similar at 35 d after pollination.The soluble protein content in the silique peel and seed decreased gradually at the mature stage,and the soluble protein content in the silique peel was higher than that in the seed at the same stage.Si+Se foliar fertilizer spraying at the seedling stage can promote the growth and yield of Brassica napus L.and can be applied in Brassica napus L.production.

Characteristics and Comparative Study of Chlorophyll-protein Complexes from Siphonous Green Algae

By mild PAGE method, 11, 11, 7 and 9 chlorophyll_protein complexes were isolated from two species of siphonous green algae (Codium fragile (Sur.) Hariot and Bryopsis corticulans Setch.), green alga (Ulothrix flacca (Dillw.) Thur.), and spinach (Spinacia oleracea Mill.), respectively. Apparent molecular weights, Chl a/b ratios, distribution of chlorophyll, absorption spectra, low temperature fluorescence spectra of these complexes were determined, and compared with one another. PSⅠ complexes of two siphonous green algae are larger in apparent molecular weight because of the attachment of relative highly aggregated LHCⅠ. Four isolated light_harvesting complexes of PSⅡ are all siphonaxanthin_Chl a/b_protein complexes, and they are not monomers and oligomers like those in higher plants. Especially, the absence of 730 nm fluorescence in PSⅠ complexes indicates a distinct structure and energy transfer pattern.

Chloroplast Composition and Structural Differences in a Chlorophyll-reduced Mutant of Oilseed Rape Seedlings

对黄化突变体Cr352 9和野生型油菜 (BrassicanapusL .) 352 9叶绿体的超微结构和组成进行了比较。与野生型相比 ,突变体Cr352 9叶片具有较少的类囊体、较少的垛叠膜区和较少的叶绿素含量。突变体的Chla/Chlb比值较高 ,是野生型的 2倍。电泳结果表明 ,突变体类囊体膜中LHCⅡ和其三聚体LHCⅡ 的含量减少。SDS_PAGE分析显示 ,LHCⅡ的脱辅基蛋白在突变体类囊体膜中明显减少。免疫印迹进一步表明 ,所有LHCⅡ组分的含量仅为野生油菜的类囊体膜的 1 / 3。突变体Cr352 9的天线系统比野生型 352 9的小。

Changes of Chorophyll protein Complexes and Photosynthetic Activities of Chloroplasts from Lotus (Nelumbo nucifera) Seeds Germinating in Light

The changes of chlorophyll_protein complexes and photosynthetic activities of chloroplast isolated from lotus ( Nelumbo nucifera Gaertn.) seeds germinating under illumination were studied. SDS PAGE analysis of chlorophyll_protein complexes showed that there was only the light harvesting chlorophyll a/b protein complex from PSⅡ (LHCⅡ) precursor in chloroplast from lotus seeds germinated for 2 to 6 days, while LHC Ⅱ 1, and the chlorophyll_protein complex of PSⅠ (CPⅠ) appeared on the 8th day of germination and PSⅡ reaction center complex appeared later. Studies on the polypeptides composition of the chloroplast revealed the following results: 1) Small amount of the 27 kD polypeptide was synthesized in invisible light; 2) The 30 kD polypeptide existed previously in the plumules of the dry seeds; 3) The amount of the 30 kD polypeptide was more than any other polypeptides before germination and decreased gradually throughout germination, while the 27 kD polypeptide changed in the opposite way; 4) In the process of germination, measurement of the electron transport rate and the fluorescence induction kinetics at room temperature showed that PSⅡ activities and efficiency of primary light energy transformation were only experimentally measurable after 7 days of germination and gradually increased afterwards. At the same time, the chl a/b ratio rose from the lower value to normal; 5) The changes of chloroplast membrane components and its functions are concomitant in concert with that of the ultrastructure of chloroplast membranes during germination, as shown in our earlier work . The results have proved again that a different developmental pathway of chloroplast is likely to exist in the lotus plumules, which might provide an important clue for N. nucifera in having an unique position in the phylogeny of the angiosperm.

Effect of Two Herbicides on the Growth of Early Seedlings of Rye(Secale cereale)

[Objective]The aim was to study the effect of herbicide on the growth of early seedlings of rye（Secale cereale）.[Method]Effect of two kinds of herbicide（Atrazine and APM）on seedling growth of rye was investigated at the physiological,biochemical and cellular level.[Result]The Atrazin significantly decreased the contents of chlorophyll a,b and soluble proteins.Rye seeds were treated with 0.01-1 mg/L Atrazine for 16 h,the contents of chlorophyll a and b decreased from 1.26（a）,0.49（b）mg/g FW（control）to 1.15（a）,0.46（b）mg/g FW（0.1 mg/L）and 0.81（a）,0.33（b）mg/g FW（1.0 mg/L）.The content of soluble protein decreased with the increasing concentration of Atrazin.Atrazin had no significant influence on the cell division and chromosome structure variation.The contents of chlorophyll a,b and soluble proteins had no significantly change under the treatment of APM,but the number of chromosome structure variation such as chromosome bridge,multipolar division cells,lagging chromosome and unequal division cells increased significantly.[Conclusion]The critical concentration of Atrazine was 0.1-1.0 mg/L and 4 mg/L of APM in rye.

Construction of a chloroplast protein interaction network and functional mining of photosynthetic proteins in Arabidopsis thaliana

Chloroplast is a typical plant cell organelle where photosynthesis takes place. In this study, a total of 1 808 chloroplast core proteins in Arabidopsis thaliana were reliably identified by combining the results of previously published studies and our own predictions. We then constructed a chloroplast protein interaction network primarily based on these core protein interactions. The network had 22 925 protein interaction pairs which involved 2 214 proteins. A total of 160 previously uncharacterized proteins were annotated in this network. The subunits of the photosynthetic complexes were modularized, and the functional relationships among photosystem Ⅰ （PSI）, photosystem Ⅱ （PSII）, light harvesting complex of photosystem Ⅰ （LHC Ⅰ） and light harvesting complex of photosystem Ⅰ （LHC Ⅱ） could be deduced from the predicted protein interactions in this network. We further confirmed an interaction between an unknown protein AT1G52220 and a photosynthetic subunit PSI-D2 by yeast two-hybrid analysis. Our chloroplast protein interaction network should be useful for functional mining of photosynthetic proteins and investigation of chloroplast-related functions at the systems biology level in Arabidopsis.

A novel chloroplast-localized protein EMB1303 is required for chloroplast development in Arabidopsis

To understand the molecular mechanisms underlying chloroplast development, we isolated and characterized the albino mutant emb1303-1 in Arabidopsis. The mutant displayed a severe dwarf phenotype with small albino rosette leaves and short roots on a synthetic medium containing sucrose. It is pigment-deficient and seedling lethal when grown in soil. Embryo development was delayed in the mutant, although seed germination was not significantly impaired. The plastids of embl303-1 were arrested in early developmental stages without the classical stack of thylakoid membrane. Genetic and molecular analyses uncovered that the EMB1303 gene encodes a novel chloroplast-localized protein. Microarray and RT-PCR analyses revealed that a number of nuclear- and plastid-encoded genes involved in photosynthesis and chloroplast biogenesis were substantially downregulated in the mutant. Moreover, the accumulation of several major chloroplast proteins was severely compromised in emb1303-1. These results suggest that EMB1303 is essential for chloroplast development.

Identification of Light-Harvesting Chlorophyll a/b-Binding Protein Genes of Zostera marina L.and Their Expression Under Different Environmental Conditions

Photosynthesis includes the collection of light and a/b-binding （LHC） proteins. In high plants, the LHC gene family constituting the light-harvesting complex ofphotosystems I and II. the transfer of solar energy using light-harvesting chlorophyll includes LHCA and LHCB sub-families, which encode proteins Zostera marina L. is a monocotyledonous angiosperm and inhab- its submerged marine environments rather than land environments. We characterized the Lhca and Lhcb gene families of Z. marina from the expressed sequence tags （EST） database. In total, 13 unigenes were annotated as ZmLhc, 6 in Lhca family and 7 in ZmLhcb family. ZmLHCA and ZmLHCB contained the conservative LHC motifs and amino acid residues binding chlorophyll. The average similarity among mature ZmLHCA and ZmLHCB was 48.91% and 48.66%, respectively, which indicated a high degree of diver- gence within ZmLHChc gene family. The reconstructed phylogenetic tree showed that the tree topology and phylogenetic relation- ship were similar to those reported in other high plants, suggesting that the Lhc genes were highly conservative and the classification of ZmLhc genes was consistent with the evolutionary position of Z. marina. Real-time reverse transcription （RT） PCR analysis showed that different members of ZmLhca and ZmLhcb responded to a stress in different expression patterns. Salinity, temperature, light intensity and light quality may affect the expression of most ZmLhca and ZmLhcb genes. Inorganic carbon concentration and acidity had no obvious effect on ZmLhca and ZmLhcb gene expression, except for ZmLhca6.

PHYSIOLOGICAL INHIBITORY EFFECT OF OCS IN ARACHIDONIC ACID-RICH PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE, CHLOROPHYTA)

Parietochloris incisa is an arachidonic acid rich snow green alga. The main physiological profiles, such as ash free dry weight (AFDW), chlorophyll, carotenoid, protein and total fatty acids (TFA), in this alga exposed to old culture supernatant (OCS) at the decline phase or its crude ethyl acetate extracts (CEAE) were investigated by using tubular photobioreactors of different diameters. Results showed that both OCS and CEAE had strong inhibitory effect on the above physiological parameters. The longer the culture was exposed to OCS and the more CEAE were added into the algal culture, the more the above physiological properties were inhibited. Arachidonic acid (AA), the dominant component of fatty acids in this alga, was also seriously inhibited with respect to total TFA, AFDW of cell mass, or culture volume, due to a probable reduction of enzymes activities catalyzing chain elongation from C18:1ω9 to AA. These results incontestably evidenced that some CEAE dissolving substances existing in OCS, like auto inhibitors, inhibited P. incisa growth through feedback. Hence, any efficient removal of auto inhibitors from algal culture to decrease their bioactivity could be good for maximal production of desired products like AA.

ANALYSIS OF EVOLUTIONARY RELATIONSHIP BETWEEN ASTASIA LONGA AND EUGLENA GRACILIS BY USING RAPD TECHNIQUE AND CLADISTIC ANALYSIS

Although both Astasia longa and Euglena gracilis belong to different genera, they share many morphological characters except that A. longa has no chloroplast. In the 1940’s, on the basis of the finding that in darkness or upon addition of some chemicals, E. gracilis would fade reversibly or irreversibly, some scholars hypothesised that A. longa evolved from E. gracilis by losing chloroplast. The authors’ use of RAPD and cladistic analyses in a study on the evolutionary relationship between A .longa and E. gracilis showed that the A. longa ’s relationship with E. gracilis was closer than that with other green euglenoids. This proves the hypothesis that A. longa evolved from E. gracilis is reasonable. The results of this study suggest that saprophytic colorless euglenoids were transformed from green euglenoids by losing their choroplasts.

Increasing the Protein Amount of Chlorella vulgaris [Chlorphyta] Strains Isolated from Different Fresh Water Ponds

Chlorella vulgaris is a single-cell, spherical green algae and one of the microalgae on which many applied studies are conducted. In the present study, five strains displaying fast and efficient reproduction were chosen among 11 C. vulgaris strains isolated from different fresh water ponds and their cell numbers and the amount of chlorophyll a, protein, lipid, cellulose and carbohydrate were examined. The main goal of the study is to investigate increasing the biochemical contents especially the protein content of C. vulgaris strains in different mediums. In the present study, cell densities were determined through cell count for five days. In parallel with cell count, their chlorophyll a content was determined. The highest cell density was observed with C. vulgaris TOH （Tourism and Hotel Management Pond） strain as 5.5 × 104 h/mL, and the chlorophyll a content as 4.3 × 102 mg/m3. The highest intracellular protein amount was determined with C. vulgaris GUH （Gazi University Rectorship Pond） （0.061 g/100 mL） and the highest lipid amount was attained with C. vulgaris UIK （Ulus Construction Well） strain as 0.019 g/100 mL. The process of increasing the intracellular protein amount in C. vulgaris GUH strain was carried out in Prat, Yagojinski and Chlorella medium. The results indicated that Chlorella medium increased the intracellular protein amount.

Oxidative stress responses of submerged macrophyte Vallisneria asiatica to different concentrations of cyanobacteria

In a 10-day aquarium experiment, this investigation examines macrophyte restoration in eutrophic Lake Taihu, the physiological effects of different plant biomass levels and of increasing natural cyanobacterial concentrations on a submerged macrophyte, Vallisneria asiatica. Cyanobacterial stress suppressed the superoxide dismutase （SOD） activity of the plant＇s leaves and induced the catalase （CAT） and peroxidase （POD） activities of its roots. The soluble protein content in V. asiatica decreased with an increase in natural cyanobacterial concentrations, whereas the malonaldehyde （MDA） increased significantly at chlorophyll a （Chl a） concentrations of 222 and 262 μg/L in water. V. asiatica adapted to the stress caused by cyanobacterial concentrations by adjusting its antioxidant defense system to remove the excessive reactive oxygen species when the algal Chl a concentration was 〉109 μg/L. Additionally, high biomass of V. asiatica （2 222 g FW/m^2） can inhibit the reproduction of cyanobacteria more significantly than low biomass （1 111 g FW/m^2）. High biomass of V. asiatica increased the oxidative stress in an individual plant when the initial Chl a concentration in the water reached 222 and 262 μg/L, as expressed by the increased MDA in leaves, compared with low biomass of K asiatica. This provides a basis for controlling cyanobacterial concentrations and V. asiatica biomass for the recovery of V. asiatica in eutrophic Lake Taihu.

Research on the Construction and Function of a Chlorophyll Degradation Recombinant Engineering Strain

In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expression vector in E. coil, the recombinant engineering strain was obtained. Afterwards, IPTG （isopropy-β-D-thiogalactopyranoside）was used to induce the goal protein, and the chlorophyllase activity of the recombinant engineering strain was measured, so as to investigate its degradation effect on the chlorophyll in the extracts of tobacco leaves. The results were as follows： （1） the amplified chlorophyllase gene At- CLH1 constructed the expression vector pET28a-AtCLH1 successfully, obtaining the recombinant engineering strain; （2） induced under 30 ℃ for 22 h, the strain could well express the recombinant protein AtCLH1 with 0.5 mmol/L IPTG, and the molecular weight was about 35 kDa; （3） the strain showed good chlorophyllase producing capability, and the activity of the produced chlorophyllase could reach up to 24.9 U/mL, which could degrade the chlorophyll in tobacco extract and had a good application prospect in improving the quality of low quality tobacco; （4） based on the results of orthogonal test, the enzyme extract from the strain was added to the tobacco leaf surface, which could make the degradation rate of chlorophyll in the tobacco leaf reach 17.06% under the temperature of 37 ℃ at the humidity of 75% for 48 h; （5） after treated by the enzyme liquid, the test tobacco showed increase in the content of aromatic substances, enhancement of tobacco fragrance quality and amount, significant decrease of offensive odor and irritation, significant improvement of agreeable aftertaste, making the overall sensory quality of the tobacco leaf significantly improved.

Deletion of a Dna K protein gene causes seedling green-revertible albino by retarding chloroplast development in rice

Green-revertible albino mutants are important sources for studying chloroplast structure, chloroplast development, chlorophyll biosynthesis, and plant photo- synthesis. In the present study, we characterized a green- revertible albino mutant gra（k）, which was obtained from the tissue-cultured rice Kitaake. The mutant gra（k） exhib- ited albino on its first three leaves. The leaf color started to turn green at the four-leaf stage. The chlorophyll contents were deeply reduced at the seedling stage, and the chloroplast development was delayed in gra（k）. The green- revertible albino （gra） phenotype of the mutant gra（k） was temperature dependent. The main agronomic traits, including plant height, tilling number per plant, seed set- ting rate, and thousand-grain weight, slightly decreased in gra（k） comparing to those in the wild-type Kitaake. Genetic analysis showed that the gra phenotype was con- trolled by a single recessive nucleic gene. By using 5,168 recessive F2 individuals derived from the cross of gra（k） × Jodan, the locus of the gene Gra（k） was delimited in a DNA region of 200 kb between the makers B-31 and P11 on chromosome 5. Sequencing analysis indicated that the three functionally annotated genes, LOC_Os05g23700, LOC_Os05g23720, and LOC_Os05g23740, were all deleted in the 200 kb region in the mutant gra（k）. Trans- genic test revealed that the gra（k） plants over-expressing LOC_Os05g23740CDS were restored to normal green as the wild-type Kitaake. Our results proved that the deletion of the DnaK protein gene LOC_Os05g23740 （encoding the chaperon protein OsHsp70CP1） led to the gra phenotype in the mutant gra（k）.