应用同工酶进行宽皮柑桔分类及其进化研究

利用聚丙烯酰胺凝胶电泳法,分析了50个宽皮柑桔生物型及5个甜橙品种的过氧化物酶、谷氨酸草酰乙酸转氨酶、四唑氧化酶、6-磷酸葡萄糖异构酶、6-磷酸葡萄糖变位酶、超氧化物歧化酶、苹果酸酶以及酯酶等8种酶系统同工酶。运用数量分类学的原理及方法,对同工酶资料进行了聚类分析。结果表明,宽皮柑桔的起源是多元的,小果类桔属于宽皮柑桔的原始类型,在进化过程中起着较为重要的作用。中国原产宽皮柑桔的代表种以道县野桔、马鼻桔或狗屎柑为佳。柑的来源有两条途径,即桔橙杂种和桔的单元演化;蕉柑是栟柑与甜橙的杂种;温州蜜柑来源于黄岩本地广桔的实生变异。     

Studies on Taxonomy and Evolution of Citrus and Its Related Genera by Isozyme Analysis

The leaf or bark tissue extracts of 108 biotypes of Citrus and its five related genera were analyzed by polyacrylamide gel electrophoresis for isozymes of peroxidase, glutamate oxaloacetate transaminase, phosphoglucose isomerase, phosphoglucose mutase, superoxide dismutase, tetrazolium oxidase, NADP+- dependent malate dehydrogenase and esterase. The inter-or intra-generic differences of isozymograms were compared. Dissimilarities of the isozyme patterns among the samples were calculated by computer based on the UPGMA method to demonstrate the phylogenetic relationship of the biotypes. There are remarkable isozymogramatic differences among the six genera. Generally speaking, each genus possesses its own unique bands. Based on the cluster analysis, the total Citrus biotypes are classified into seven groups, i.e. Honghe papeda, Mauritius papeda, Pummelo, Ichang papeda, Citron, Lemon-lime and Mandarin-orange. Ichang papeda is suggested to be the third subgenus of the genus Citrus. The Fuming trifoliata orange is accepted as a new species of Poncirus-P. polyandra S. Q. Ding. It might be the evolutionary bridge between Poncirus and Citrus, and one of the “missing links” as suggested by W. T. Swingle. Microacrumen (small- fruited mandarin) is the primitive form of the mandarins. It seems better to consid er“ Horse-nose mandarin ”or “Xipigoushigan mandarin” as the typic mandarin originated in China. Euacrumen (large-fruited mandarin) might have arised through two ways. One is hybridization of Microacrumen and C. sinensis, and the other is just the variation of Microacrumen. Ancestors of some biotypes are inferred based on isozyme data. Fortunella obovata Tan. may be a hybrid of Fortunella and Citrus. C. unshiu Marc. arose from the nucellar mutation of“Huangyanbendiguangju mandarin”( C. nobilis Lour.) in Huangyan of Zhejiang Province. C. junos Sieb. came from hybridization of Ichang papeda and mandarin. C. grandis Osbeck and mandarin may have been involved in the origin of C. aurantium L.. C. limonia Osbeck may be a hybrid between C. medica L. and mandarin. The evolutionary trend of citrus fruit trees is proposed, anda tentative phylogenetic diagram is drawn.     

A Study on Glutamate Oxaloacetate Transaminase Isozymes of Citrus Germplasm Resources

The glutamate oxaloacetate transaminase isozymes (GOT) extracted from 125 biotypes of Citrus and its relatives, Fortunella, Poncirus and Microcitrus were analyzed using polyacrylamide gel electrophoresis to investigate the taxonomic relationships among citrus plants. Besides all the isozymes reported before, two new bands were detected and designated their putative alleles B and C in GOT-l. Among pummelo cultivars wide variations were found. Most of the mandarins were identical, having SS for GOI-1 and MM for GOT-2 except for Zou-Pi-Gan and Yao-Gan, which both had FS at GOT-1 and might be hybrids. This finding suggests that all of the mandarins may have originated from a common ancestor. Sour orange biotypes showed a considerable variation in GOT isozymes. Most of the sour oranges in China were assumed to be hybrids between pummelo and mandarin based on GOT isozyme patterns, but two biotypes, Xiao-Hong-Cheng and Zhu-lan, had FS at GOT-1 and MB at GOT-2, which strongly suggests that they be hybrids of pummelo and Ichang papeda because B allele of GOT-2 occurs only in Ichang papeda and its close relatives Yuzu and Ichang lemon. From this study Yuzu is assumed to be derived from hybridizationof Ichan, papeda and mandarin.     

锌胁迫对“不知火”和“椪柑”的生理指标及锌分布的影响

采用砂基培养法,研究了0、0.05和0.5 mg·L^-1Zn²⁺（0.05 mg·L^-1为对照,0.5 mg·L^-1为锌过量,0 mg·L^-1为锌缺乏）处理下“不知火”和“椪柑”叶片的一些生理指标及不同部位锌含量的变化。结果表明：1）缺锌处理的“不知火”叶绿体色素含量和叶面积均显著低于其对照和锌过量处理;锌过量处理的“椪柑”叶绿体色素含量和叶面积均显著低于对照。2) 3个锌浓度处理间,“不知火”叶片POD、“椪柑”叶片CAT活性无显著性差异,“不知火”叶片CAT、SOD活性随着锌浓度的升高而升高,锌缺乏处理下“椪柑”叶片POD、CAT、SOD活性均显著地高于“不知火”,而锌过量时“不知火”叶片SOD活性显著高于“椪柑”;锌胁迫下“不知火”和“椪柑”叶片MDA含量均显著高于对照,锌过量时“椪柑”叶片MDA含量显著高于“不知火”。3）2品种柑橘不同部位的锌含量随着锌处理浓度的升高而升高,在相同浓度的锌处理下“椪柑”叶锌含量显著高于其他部位及“不知火”叶锌含量,“不知火”上部叶锌含量显著高于其下部叶。     

Cold treatment of Ceratitis capitata (Diptera: Tephritidae) in oranges using a larval endpoint

South Africa currently exports fresh citrus (Citrus spp.) fruit to Japan using an in-transit cold treatment protocol of 14 d or 12 d at temperatures <0 degrees C for treatment of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) in 'Clementine' mandarins (Citrus reticulata Blanco) and other citrus types, respectively. To reduce the risk of chilling injury with this treatment, research was conducted with temperatures >0 degrees C. Earlier South African research had shown that young (6-d-old) larvae were slightly more tolerant of cold treatment and that there were no significant differences between cold tolerance of these larvae in different citrus types [oranges, Citrus sinensis (L.) Osbeck; grapefruits, Citrus paradisi Macfad.; lemons, Citrus limon (L.) Burm.f.; and mandarins). Due to their ready availability, 'Valencia' oranges were used in this study. When 62,492 larvae in total were treated in three replicates at a mean temperature of 1.5 degrees C for 16 d, there were three larval survivors. The trial was therefore repeated with oranges using a 16-d period at a mean temperature of 1.0 degrees C and a mean of 1.4 degrees C for the hourly maximum probe readings. Three replicates were again conducted and the resultant mean mortality in the control was 8.1% of 21,801 larvae, whereas the cold treatment mortality was 100% of 71,756 larvae. This treatment at a mean temperature of 1 degree C exceeded the Japanese confidence level requirement and also exceeded the Probit-9 mortality level, but not at a confidence level of 95%. These data support the establishment of a treatment protocol of 16 d at temperatures <1.4 degrees C, commencing once all fruit pulp probes reach a temperature of 1 degree C or lower.     

不同砧木椪柑氮素营养的研究

研究结果表明,不同砧穗组合椪柑的氮素营养存在差异。半矮化枳砧椪柑,无论是枳砧根系或是枳砧椪柑叶片,在年周期中(或一定阶段),其全氮、硝态氮含量和硝酸还原酶活性多处于较高水平,且呈现出高于两种乔砧(椪柑、福桔)椪柑组合。试验表明,枳砧根系的较高氮素水平,促进了接穗椪柑叶片氮素水平的提高,作者认为,半矮化枳砧椪柑植株氮素营养的较高水平与碳索同化水平相一致,是其早结高产的一个重要因素。     

应用过氧化物酶同工酶鉴定山茶属植物杂种F1代的研究

对4个杂交组合的12个杂种F1代植株与亲本进行了形态学比较和过氧化物酶同工酶分析,结果表明以金花茶为父本,云南山茶或云南野山茶为母本杂交产生的11个杂种的幼枝、叶的特征均与母本相似,H-86-1-1的花与母本一致,H-87-2-2的花兼有父母本的特征;H-78-1-1的幼枝、叶的特征与父本相似,其花也兼有父母本的特征。有9株的酶谱为“互补型酶带”,且都出现了杂种带,为真正的杂种;其余2株的酶谱与母本一致,与其形态特征表现出一定的相关性,可能为非真正的杂种。同一个杂交组合产生的杂种,酶谱却有差异,金花茶最为特征的一条带(Rf=0.813)在其所有11个杂种F1代植株中都未表达。     

柑桔类的细胞分类学研究 Ⅰ.柑桔属30个分类群的核型及进化

作者研究了柑桔属30个分类群的核型及进化。结果表明:染色体数目为2n=2x=18,属小染色体植物,核型由中部着丝粒染色体(m)和近中部着丝粒染色体(sm)组成,具2—4个连接于短臂上的随体,一些分类群的部分同源染色体上存在长度和臂比值不同程度的杂合现象,核型均属2A型。比较随体及与之相连短臂,其随体分为大随体(LS)中随体(MS)和小随体(SS)三类,随体的多样性可作为探讨该属物种的起源、系统演化的一个遗传标志。作者赞同把柚(Citrus grandis)作为柑桔属的一个基本种,而宽皮柑桔(C.reticulata)核型变异较大,还待进一步分析;推测红黎檬(C.limonia)、葡萄柚(C.paradisi)、甜橙(C.sinensis)、酸橙(C.aurantium)、香园(C.wilsonii)、三宝柑(C.sulcata)、部分宽皮柑桔(C.reticulata)为杂种起源。该属核型由不对称向对称进化是与形态及孢粉的进化趋势一致的。     

The pH induced modification of the electrophoretic mobilities of horseradish peroxidase isozymes

Isozymes of horseradish peroxidase may be generated from preexisting forms of the enzyme by incubation at 4 °C in solutions with pH's of 7 or higher. Isozymes generated in this manner express an apparent net increase in negative charge compared to the original form of the enzymes. This is evidenced by an increase in anodic electrophoretic mobility and a decrease in isoelectric point. The generation of new isozymes of peroxidase by such treatment alters the isozyme distribution pattern considerably, but there is no net change in total peroxidase activity present in the extract if pH's of 10.0 or lower are used. The generated peroxidase isozymes are formed irreversibly; neither retitration of extracts to a lower pH nor heat treatment will restore the original peroxidase isozymes.     

Multiple lignin peroxidases of Phanerochaete chrysosporium INA-12.

Nine proteins with lignin peroxidase activity were separated from cultures of Phanerochaete chrysosporium INA-12 in glycerol as carbon source and non-nitrogen limited. Four lignin peroxidase isozymes (4, 5, 8, 9) were purified and characterized. Although differences in kinetic parameters could be shown, antibody reaction showed homology between isozymes. However, thermal stability studied, peptide mapping results, and N-terminal sequence analyses established a higher degree of homology between isozymes 4/5 and 8/9 types. Protein characterization and kinetic data indicate that lignin peroxidase isozymes 4, 5, 8, and 9 differ from described isozymes in strain BKM. The higher specific activity of lignin peroxidase isozymes in cultures with glycerol than in nitrogen-starved cultures accounts for the higher lignin peroxidase activity obtained in these conditions.     

杉木不同无性系过氧化物酶同工酶的研究

本文对杉木（Ｃｕｎｎｉｎｇｈａｍｉａｌａｎｃｅｌｏａｔａ（Ｌａｍｂ．）Ｈｏｏｋ．）不同无性系及其不同株龄的叶和不同发育时期的雄球花进行了过氧化物酶同工酶检测。结果表明：杉木４１４号无性系叶和雄球花的过氧化物酶同工酶酶谱与其它无性系都具明显差异;同－无性系叶与雄球花的过氧化物酶同工酶酶谱存在显著差异;不同发育时期的雄球花的过氧化物酶同工酶存在顺序表达。作者认为：过氧化物酶同工酶作为种内分类的鉴定性状较为合适,而科、属、种的分类依据则应更侧重于形态特征。根据杉木４１４号无性系的过氧化物酶同工酶的特异性及其短叶、叶端钝圆和多雄花等特点,可以把它定为杉科杉属杉木物种的一个变型：多雄花杉木。     

Inhibitors of platelet lipoxygenase from Ponkan fruit

An activity-guided separation for inhibitors of rat platelet 12-lipoxygenase led to the isolation of two compounds, 4-O-feruloyl-5-O-caffeoylquinic acid (IC50; 5.5 microM) and methyl 4-O-feruloyl-5-O-caffeoylquinate (IC50; 1.9 microM) from the peel of Ponkan fruit (Citrus reticulata). The complete structure of each phenolic ester was determined by NMR spectroscopy [1H and 13C NMR spectra, 1H-1H correlation spectroscopy (COSY), 1H-detected heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC) spectroscopies] and other spectral methods.     

Segregation of the isozymes of flax genotrophs

The segregation of isozymes of peroxidase and acid phosphatase in progenies of crosses between large (L) and small (S and L₆) flax genotrophs has been determined. The peroxidase isozymes segregated as expected on a simple Mendelian model with a dominant and a recessive allele and with the L genotroph being a homozygous dominant. All the peroxidase isozymes which differed segregated together, so the isozymes are controlled by either a single locus or closely linked loci. The acid phosphatase isozymes in the F₁ were all L type, but the segregations observed in the F₂ were not always consistent with a simple Mendelian model.     

Studies on the Esterase Isozymes in the New Lines of Progeny of the F1 Hybrid Rice

The new lines Nanhua 5, Nanhua 11, Shanyou 39, and Shanyou 59, are high-yield lines derived from the F1 hybrid rice, Nanyou 2 and Shanyou 2 by means of tissue culture and selec- tion. Five isozymes, esterase, peroxidase, ATP-ase, malate dehydrogenase and glulamate dehydrogenase from the new lines, the F1 hybrid rices and their parents, were analyzed by starch and polyacrylamid gel electrophoresis. There are no difference in zymogrames of ATPase, malate dehydrogenase, glulamate dehydrogenase, between the F1 hybrid and its parents. But the F1 hybrid rice contains complemental bands of anodal esterase EA2, EA3 and cathodal esterase EC1, EC2, EC3, EC4, The esterase zymogram of seeds of Nanhua 5, Nanhua 11 and Shanyou 39, Shanyou 59 could be observed in the progeny of the F2 hybrid rice. The results indicated that the new lines would be possibly derived from the progeny of the F1 hybrid by means of tissue culture and selection.     

Oranges and lemons: clues to the taxonomy of Citrus from molecular markers

Go into any grocery store and one is confronted with an array of Citrus fruit: oranges, grapefruit, mandarins (tangerines), lemons and limes. This is rich bounty for the shopper, but taxonomists are perplexed as to how to classify the various kinds of Citrus that have existed since antiquity. Now, thanks to new genetic and molecular biological techniques, the relationships between these fruit are being unraveled and show that there are probably only three true species.     

Peroxidase and leucine-aminopeptidase in diploidMedicago species closely related to alfalfa: Multiple gene loci,multiple allelism,and linkage

Summary The genetics of peroxidase and leucine-aminopeptidase isozymes was studied utilizing starch gel electrophoresis in the diploidsMedicago sativa L. (M. coerulea Less.) andM. falcata L. Three anodal and one cathodal sets of peroxidase isozymes identify four linked loci. In addition, two anodal sets of leucine-aminopeptidase isozymes identify two loci that may be linked. The allozymes at each of the loci segregated as expected for monomeric enzymes. However in several crosses there were deficiencies in the number of progeny in particular genotypic classes. This could result from the segregation of recessive deleterious genes linked to some of the allozyme alleles. This is the first report of multiple loci and multiple alleles determining isozymes inMedicago. Supported by grants from the Alberta Research Council (No. D1B02 to R.C. von Borstel) and the Computer Use and Policy Committee, University of Alberta     

The Genome Sequence of an Isolate of Indian Citrus Ringspot Virus Infecting the Sweet Orange in India

Whole-genome sequencing of an isolate of Mandarivirus infecting the sweet orange [Citrus sinensis (L) Blanco] in the western part of India (Pune) was done. The single-stranded positive-sense RNA genome of Indian citrus ringspot virus (ICRSV) Pune has 7,560 nucleotides (nt), excluding a poly(A) tail, comprised of 27.98% (2,115 nt) A, 32.12% (2,428 nt) C, 19.68% (1,488 nt) G, and 20.22% (1,529 nt) T residues. The genome, organized into six open reading frames (ORFs), shares 97.7% sequence identity with the complete genome of the ICRSV K1 isolate ({"type":"entrez-nucleotide","attrs":{"text":"AF406744.1","term_id":"15420945","term_text":"AF406744.1"}}AF406744.1) infecting the kinnow (Citrus reticulate Blanco, a hybrid between King and Willow mandarins) in north India. The ICRSV Pune genome formed a complex secondary structure with a large number of unpaired cytosine-rich regions, and recombination analysis highlighted potential recombination in the ICRSV genome.     

Carbohydrate and ethylene levels related to fruitlet drop through abscission zone A in citrus

Citrus fruits have two abscission zones (AZ), named A (in the pedicel) and C (in the calyx). Early fruitlet abscission takes place exclusively through AZ A, while at June drop it is progressively inactivated and AZ C begins to operate. In previous work, it has been demonstrated that carbohydrate and ethylene regulate fruit drop through abscission zone C. In this paper, we have analysed the effect of these two factors in developing fruitlets of Satsuma mandarins (Citrus unshiu [Mak.] Marc.) cv. Okitsu to elucidate their involvement on abscission through AZ A. The data indicated that ACC content and ethylene production of fruitlets paralleled abscission rates. Sucrose supplementation increased fruit set, although did not counteract the abscising effect induced by ACC. Branch girdling of terminal fruitlets carrying several leaves significatively reduced ethylene production and abscission rates, and increased sugar content. Pedicel girdling showed the opposite. Taken together, the results revealed that the carbohydrate content may be a biochemical signal involved in the mechanisms controlling abscission through AZ A. The evidence also showed parallelisms between ethylene and its activation. As the induction of higher ethylene levels after the period of AZ A activity, however, was not able to promote fruit drop, it is also concluded that solely ethylene is not sufficient to activate abscission.     

Altered levels of antioxidant enzymes associated with two mutations in tomato

Activity of peroxidase, superoxide dismutase and catalase were examined in leaves, stems and roots of olivacea ( oli ) and monstrosa ( mon ) mutants of Lycopersicon esculentum Mill. The extent of the difference between the pattern of oxidative enzyme activities of the wild type (wt) and the mutants was determined. The high peroxidase activity during the developmental stages of the leaves and stems of oli and mon phenotypes is associated with high levels of 4 anodic peroxidases in leaves and of two isozymes in the stem. Leaves of oli exhibit higher activity of the cathodic peroxidase C2, while both mutations have a marked increase of peroxidase C1 in stems. A positive relation between high peroxidase activity and oxidative stress damage was found: in chilling experiments at 5°C, peroxidase level in mutants and wt leaves was negatively correlated with electrolyte leakage. Superoxide dismutase (SOD) activity rises in oli stems around flowering time due to the high activity of the chloroplast forms SOD-1 and SOD-2. Catalases (CAT) were detectable only in early stages of plant development; CAT-2 was nearly absent in wild type tissues but well represented in mon and oli. The oli and mon mutations may affect critical steps of a regulatory pathway controlling various classes of oxidative enzymes in tomato.     

A Comparison of Stem Peroxidases in Bush and Vine Forms of Squash (Cucurbita maxima Duch.and C. pepo L.)

Total peroxidase activities and peroxidase isozymes were comparedamong 10 bush- and vine-squash cultivars of Cucurbita maximaDuch. and C. pepo (L.) Several qualitative variations of isozymeswere detected between C. maxima and C. pepo, and some differenceswere evident among varieties of C. pepo. There were no qualitativedifferences between genetically similar bush and vine strains.Quantitative differences were difficult to resolve, althoughit did appear that in varieties of C. maxima exhibiting lowlevels of peroxidase activity, isozymes C₃, C₄, and C₆ wereless intense. Depending upon the cultivars compared, total peroxidase activityin bush forms was either higher or lower than, or the same astheir vine counterparts. Since the major bush genes in the varietiesstudied are believed to be allelic, the results indicate thatthe over-all genotype of squash varieties can influence therelative expression of peroxidase activity between bush andvine forms.