Analysis of Amylase and Superoxide Dismutase Isozymes During the Germination Process of Emmenopterys henryi Oliv Seeds

[ Objective] The study was to understand the changes of amylase（AMY） and superoxide dismutase（SOD） isozymes during the ger- mination process of Emmenopterys henryi Oliv seeds. [ Metbod] By employing polyacrylamide gel electrophoresis method, the expressions of AMY and SOD isozymes during seed germination process were analyzed. ~ Result] The main AMY bands remained strong during the whole peri- od and a new band A2 appeared in the middle and late period of seed germination. Some new SOD bands occurred at the early stage, then be- came weak or disappeared in the middle period, and band S6 became intense in the late peried. [ Conclusion.] The expression of AMY and SOD isozyme gene has temporal difference during germination of E. henryi Oliv seeds.

Effects of Hg^(2+) on Isozymes of Peroxidase,Catalase and Superoxide Dismutase in Wheat Seedlings

[Objective] This work was aimed to explore the mechanism of Hg2+ toxicity on plants.[Method]Activities of peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)were investigated in wheat(Triticum aestivum L.)seedlings under Hg2+ stress at different concentrations.[Result]① There were no obvious effects on the growth of seedlings when the concentration of Hg2+ was lower than 0.10 mmol/L.However,toxic effects on the growth of seedling were observed when the concentration of Hg2+ was higher than 0.10 mmol/L.② Different tissues showed different resistant ability in response to Hg2+ stress.The leaves and roots of wheat seedlings were more insensitive to Hg2+ toxicity.③ CAT was more sensitive to Hg2+ stress compared to POD and SOD.[Conclusion]The toxic effect was related to the concentration of Hg2+(0.10 mmol/L).The higher concentration of Hg2+ could affect the expression of POD,CAT,and SOD isozymes in the leaves,roots of wheat seedlings and germinated seeds,which further affect the normal metabolism of membrane lipid and inhibit the growth of wheat seedlings at last.

Electrophoretic Analysis of Isozymes and Discussion about Species Differentiation in Three Species of Genus Gymnocypris

By using the method of electrophoresis,three isozymes (lactate dehydrogenase,malate dehydrogenase and esterase) of three species of genus Gymnocypris were described and analyzed from North Tibet in this paper. The results showed that all three isozymes presented interspecific difference and distinct differentiation among individuals in the same population,and there was no electrophorectic difference between males and females. Analysis of relationships among three naked carps indicated a high degree of similarity between G. selincuoensis and G. cuoensis ,whereas low degree between G. selincuoensis and G. namensis . Furthermore,three isozymes presented expression of null alleles,and the duplicate genes of LDH A 2,LDH B 2,s MDH A 2 and m MDH B 2 also expressed in some individuals. Compared to other tetraploid fishes,three naked carps retained more functional duplicate genes and null alleles. This suggests fishes of genus Gymnocypris are at the early stage of evolution after polyploidization than that of fishes of Catostomidae,it directly related to the later time of schizothoracine fishes originate as well as severe environment.

Comparative Study of Immunological Properties on Glutamine Synthetase Isozymes in Rice Plants

In rice (Oryza sativa L.) roots two glutamine synthetase (GS) isozymes, GSra and GSrb, were identified recently in the author's experiments, but the homology of both GSra and GSrb as well as their localization in the rice roots are unclear. In the present study, the purified GSra and GSrb from rice roots were used to immunize rabbits to obtain the respective antibodies. The immunodiffusion and immunoblotting experiments showed that the antibody against GSra or GSrb was specific for GS and its isozymes. The immunoprecipitation test indicated that the antibody of GSra or GSrb not only recognized its respective antigen, but also well recognized each other's antigen. GSra or GSrb antibody recognized also better cytosolic GS1 of rice leaves, but the recognization for chloroplast GS2 from rice or spinach (Spinacia oleracea Mill.) leaves was weaker. Our results indicate that GSra and GSrb from rice roots are quite similar in antigenicity and are extremely similar proteins and that both GSra and GSrb may also be a form of cytosolic GS just as the cytosolic GS1 of rice leaves.

Isozyme Phenotypic Difference of Varicorhinus macrolepis Malic Enzyme

By using polyacrylamide gel electrophoresis and biochemical straining method, malic enzyme isozyme in 10 organs of male or female Varicorhinus macrolepis were analyzed. The results showed that malic enzyme exists in heart, gonads, eyes and gills tissues in both female and male V. macrolepis. There was only one ME-2 in heart and ME-1 in eye of female fish, while there were ME-1 and ME-2 in heart and ME-2 in eyes of male fish. Ovary contained ME-1 and ME-2, while spermary only contained ME-1. In kidney and spleen tissues, malic enzyme was found only in female fish, while in muscle, malic enzyme existed only in male fish. The phenotypic of malic enzyme had obvious differences no matter in the same organ of different sexes in V. macrolepis or different organs in the same sex. But there were no differences among different individuals of the same sex. The results will provide basic data for developmental genetics, breed improvement and directed breeding of V. macrolepis, and lay a foundation for its development and protection.

6 Kinds of Isozymes after Long-term Subculture of Emmenopterys henryi Oliv.

[Objective] The aim was to study the changes of zymography in 6 kinds of isozymes after long-term subculture of Emmenopterys henryi Oliv.. [Method] Non-denaturing polyacrylamide gel electrophoresis was used to analyze isozyme patterns such as esterase (EST),acid phosphatase (ACP),ATP enzyme (ATPase),amylase (AMY),superoxide dismutase (SOD) and peroxidase (POD) in long-term subculture callus of Emmenopterys henryi Oliv. [Result] The research showed that there were differences among the 6 kinds of isozymes in embryogenic callus and non-embryogenic callus of Emmenopterys henryi Oliv.,and both levels could be taken as the basis for identification,the EST,ACP,and POD of non-embryogenic callus were significantly higher than embryogenic callus. The browning of non-embryogenic callus was non-level in the AMY,SOD and POD isozymes when was compared with normal non-embryogenic callus,while the EST,ACP and ATPase isozymes decreased; When the browning of embryogenic callus was contrasted with normal embryogenic callus,EST isozyme increased and the other 5 kinds of enzymes decreased. [Conclusion] The study provided theoretical basis for research morphological difference and browning of long-term tissue culture of Emmenopterys henryi Oliv..

Genetic Analysis of Five Porphyra Species by Isozyme Markers

Vertical polyamide gel electrophoresis was used to investigate isozyme polymorphisms among different isolates （including wild and cultivated） of Porphyra katadai, Porphyra oligospermatangia, Porphyra yezoensis, Porphyra haitanensis, and a hybridize species （Porphyra yezoensis x Porphyra haitanensis） sampled from China. Whereafter, the analyses of probable minimum loci numbers, observed alleles sum, genetic diversity, and unweighted pair-group arithmetic average （UPGMA） cluster were carded out. After initial activity and resolution testing of bands of 23 enzymes, 6 of them （MDH, ME, LDH, GDH, IDH and G-6-PDH） were proved to be appropriate for analysis of the full sample set. The probable minimum numbers of loci and alleles analyses showed that the five species of Porphyra had an extraordinary consistent result in ME loci and alleles. However, P. katadai and P. oligospermatangia differed from other three species of Porphyra in LDH and GDH loci and alleles. P. katadai was independent in the analyses of MDH and P. oligosperTnatangia and P. haitanensis differed from other three species in IDH analyses. Moreover, P. yezoensis and P. haitanensis were apart from other three species in G-6-PDH analysis. Taking one with another, P. katadai was relatively separated in the probable minimum numbers of loci and alleles analyses. The results indicated that the genetic variation among the five Porphyra species was limited with a genetic identity of 0.7550. The hybridize species （P. yezoensis x P. haitanensis） seemed to be high homologue with P. oligospermatangia, unexpectedly got relatively lower average genetic identities with both P. yezoensis and P. haitanensis. The 4 strains of P. yezoensis were relatively divergent with an average genetic identity of 0.7428, and P. katadai presented the most differentiated, compared with other species, which consistented with the result summarized in the probable minimum numbers of loci and alleles analyses.

Isozyme Analysis of Jin Silver Carp (Hypophthalmichthys molitrix Var Jin)

[Objective] The aim was to carry out isozyme analysis of jin silver carp (Hypophthalmichthys molitrix Var Jing). [Method] The isozyme of AAT, EST, α-GPD, GPI, IDH, LDH, MDH, ME, PGM and PROT of muscles and liver in two populations of the silver carp (Hypophthalmichthys molitrix): Jin silver carp (a breed through selective breeding) and artificially propagated population bought from Jingzhou city, Hubei Province were examined by horizontal starch gel electrophoresis. [Result] Eighteen loci were observed in two populations. Two loci of GPI* and PGM* in Jing silver carp population and the locus of GPI* in Jingzhou population were polymorphic. The proportions of polymorphic loci (maximum allele frequency≤0.99) of Jing silver carp and Jingzhou populations were 11.11% and 5.56% respectively, expected heterozygosity were 0.015 0 and 0.001 1 respectively. The Nei’s genetic distances were 0.000 59 between two populations. The result of chi-square test of the GPI* gene in two populations showed that their genetic structure has very significant difference. [Conclusion] This study provided a theoretical basis for large-scale extension of Jing silver carp.

Comparison on Esterase Isozymes and Peroxidase Isozymes of Black Worm Tenebrio molitor Linnaeus at Different Developmental Stages

[ Objective ] To respectively compare esterase isozymes and peroxidase isozymes among black worm Tenebrio molitor Linnaeus （ Tenebno molitor L. ） at different developmental stages and thus provide a theoretical basis for species identification and large-scale farming. [ Method] The esterase isozymes and peroxidase isozymes of black worm Tenebno molitor L. at different developmental stages were respectively analyzed by polyacrylamide gel electrophoresis. [ Result] Both esterase isozymes and peroxidase isozymes were different among black worm Tenebrio molitor L. at developmental stages, larvae in different instars or between the male adults and the female adults. [ Conclusion] The esterase isozymes, peroxidase isozymes and physiological characteristics of black worm Tenebrio molitor L. varies with developmental stages.

Isozymes Analysis of the Golden Cuttlefish Sepia esculenta (Cephalopoda: Sepiidae)

Thirty nine isozymes in four tissues (mantle muscle, buccal bulb muscle, eye and liver) of Sepia esculenta were screened for enzymatic analysis using starch gel electrophoretic technique. Eighteen enzymes (G3PDH, LDH, MDH, MEP, IDHP, PGDH, GRS, NP, AAT, CK, AK, EST, ALP, ACP, FBP, MPI, GPI and PGM) show strong activities and good convergence in zymogram. They are proved to be suitable genetic markers in Sepia esculenta. Among the tissues used, mantle muscle is the best for electrophoretic analysis of isozymes. Eye and liver are fairly good for some special enzymes, such as LDH, EST, MPI, etc. Twenty six loci are detected. The proportion of polymorphic loci is 0.115 in the Qingdao sample and 0.153 in the Rizhao sample ( P < 0.99 ). The mean values of the observed and expected heterozygosity per locus of Qingdao sample are 0.016 and 0.017 , while those of the Rizhao sample are 0.023 and 0.025 respectively.

Isozyme Analysis of Spotted Halibut Verasper variegatus Temminck et Schlegel

To investigate the tissue-specificities of isozymes and the genetic structureof wild spotted halibut ( Verasper variegatus) population, horizontal starch gel electrophoresiswas performed on 45 individuals collected in part of the Yellow Sea. The performances of 17 isozymesin 8 kinds of tissues or organs were screened preliminarily in a TC-7.0 buffer system. The resultsshowed that the screened isozymes displayed remarkable tissue-specificities. Finally, 14 enzymes(AAT, ADH, EST, OPI, G3PDH, IDHP, LAP, LDH, MDH, MPI, PGDH, PGM, SDH and SOD) and 4 kinds of tissues(eye, skeleton muscle, liver and heart) were selected for genetic analysis. Fourteen isozymes areencoded by 20 loci, and 9 of them are polymorphic. The polymorphic loci are AAT-1~*, GPI-2~*,G3PDH~*, IDHP-1~*, LDH~*, MPI~*, PGM-1~*. PGM-2~* and SDH~*, and the proportion of polymorphic lociis 0.4500 (P_(0.99)) ? The mean values of observed and expected heterozygosities are 0.0278and 0.0265, respectively and the average effective number of alleles is 1.0675.

Effect of biliary obstruction and internal biliary drainage on hepatic cytochrome P450 isozymes in rats

AIM: To investigate the total cytochrome P450 (CYP) content, microsomal mixed-function oxidase (MFO) activity, and expression of mRNAs for various CYP isozymes in a simple rat model of reversible obstructive jaundice. METHODS: Obstructive jaundice was created in male rats by causing bile duct obstruction with polyester tape. In another group of rats, bile duct obstruction was followed by internal biliary drainage after releasing the tape. The expression of various CYP isozyme mRNAs was semi-quantitatively assessed by competitive RT- PCR. RESULTS: The total CYP content and microsomal MFO activity showed a significant decrease after biliary obstruction, but returned to respective control levels after biliary drainage. A marked reduction in the expression of CYP1A2, 2B1/2, 2C11, 2E1, 3A1, and 3A2 mRNA was detected during biliary obstruction, while expression increased significantly toward the control level after biliary drainage. Although expression of CYP4A1 mRNA showed no reduction during biliary obstruction, it still increased significantly after biliary drainage. CONCLUSION: These results suggest that not only obstructive jaundice, but also the subsequent internal biliary drainage may affect regulatory medications of the synthesis of individual CYP isozymes differently.

Isozyme Analysis on the Populations of Ruditapes philippinarum

Horizontal starch gel electrophoresis was used to investigate the genetic structure of six populations of Ruditapes philippinarum in the coast of China. Seven enzymes revealed eleven putative loci, and seven of them were polymorphic, PGM^＊, MDH-1^ ＊, MDH-3^＊, LAP-1^＊, LAP-2^＊, MPI-1^＊ and MPI-2^＊. The proportions of polymorphic loci of R. philippinarum populations varied from 0.454 5 to 0.636 4. The values of observed and expected heterozygosities were from 0.039 4 to 0.154 5 and from 0.111 1 to 0.238 5, respectively. Nei＇s genetic distance varied from 0.003 3 to 0.025 3 with an average of 0.013 5. It is suggested that the genetic diversity of R. philippinarum was high.

Effects of 1 — 4℃ low temperature treatment on seedling growth and POD isozymes in higher plants

The effects of iow temperature treatment on seedling growth and POD isozymeswere studied using hyacinth bean (Dolichos tablab L. )and Soybean[Glycine max(L. )Merr. ]culti-vars as experimental materials. Their seedtings were exposured to 1 - 4℃ low temperature for 4 days to inhibit growth. The changes of POD isozymes in roots and seedlings were observed after seedlings were exposured to 1 - 4℃ for 4-8 days. There was a significant linear relation between inhibition of seedling growth and change of POD isozymes in these two kinds of plants.

Phylogenetic Relationship among Ten Species of Riptortus linearis in Coreinae (Hemiptera∶Coreidae) Based on EST Isozyme

[ Objective ] The paper was to explore the phylogenetic relationship among 10 species of insects in Cletus, Hygia, Acanthocoris, Coreus and Homoeo- cerus in Coreinae. [ Method] Using vertical slab PAGE, EST isozymes of 10 species of Riptortus linearis in Coreinae were studied. [ Result] Cluster analysis showed that the difference between 5 genera of Coreinae were greater than intraspecific differences. The genus Homoeocerus had more distant relationship with the other 4 genera of Coreinae, while it had cleser relationship with Mictinae. [ Conclusion] EST isozymes of 10 species of the subfamily were clear with strong pely- morphic and good stability, which could be used for insect identification of Coreinae and phylogenetic study of species. Combined with morphological characters and variation condition of Cyt b gene sequence, it is suggested that Homoeocerus should be separated from Coreinae and classified into Mictinae.

Antioxidant and isozyme features of two strains of Laminaria japonica (Phaeophyceae)

Healthy sporophytes of two gametophyte mutants of Laminariajaponica with different heat resistances： kelp 901 （901, with comparatively stronger heat-resistance） and Rongcheng No. I （RC, sensitive to heat stress）, were respectively collected during October to December 2002 from Yantai and Rongcheng Sea Farm in the Shandong Peninsula of China. The contents of some biochemical materials and antioxidant capacity were analyzed under controlled laboratory conditions to identify if there is any relation between the overall antioxidant capacity and the heat-resistance in L. japonica and to understand possible mechanism of heat-resistance. Results show that： （1） the overall antioxidant capacity in healthy sporophyte of 901, such as vitamin E, polyphenol, and ascorbic acid contents and the enzymatic activity of SOD, POD, CAT, Gpx, PPO, and PAL, were not always higher than that of RC under controlled laboratory conditions, and no significance （P〉0.05） was shown in total antioxidant capacity （T-AOC） in 901 and RC. Result suggested that the difference in antioxidant capacity was not a decisive factor for different heat-resistances in L. japonica; （2） the simultaneous assay on isozymes was carried out using vertical polyacrylamide gel electrophoresis （PAGE）. Considerable differences in peroxide （PRX）, malate dehydrogenase （MDH）, malic enzyme （ME）, polyphenol oxidase （PPO） and glutamate dehydrogenase （GDH） were obtained in 901 and RC from either the band number, relative mobility （Rf）, or staining intensity, and ME could be used as an indicator to distinguish healthy sporophyte of 901 and RC under controlled laboratory conditions.

Molecular identification of sex in Hippophae rhamnoides L. using isozyme and RAPD markers

In many dioecious plants, gender affects economic value, breeding schemes and opportunities for commercial harvests. Hippophae rhamnoides L. is a dioecious plant species in which female genotypes are commercially preferred over male genotypes. Its berries have rich medicinal, nutritional and pharmaceutical properties because of their large amounts of vitamins, essential oils, proteins, fatty acids, free amino acids and flavanoids. Primary limitation for breeding H. rhamnoides L. is its dioecious nature, since gender cannot be identified by traditional methods. Therefore, some reliable and quick methods need to be developed. This commu- nication deals with the development of isozyme and RAPD markers for early sex identification in this dioecious tree. The isozyme analysis was conducted with four enzyme systems, viz. peroxidase, esterase, malate dehydrogenase and catalase. The peroxidase enzyme system produced a female specific sex marker, which successfully differentiated between the staminate and pistillate geno- types ofH. rhamnoides L. Thirty five random decamer primers were used in our study and one male sex linked marker was identified. OPD-20 （5＇-ACTTCGCCAC-3＇） displayed a band at 911 bp that expressed polymorphism between male and female genotypes. The staminate and pistillate genotypes could be distinguished using RAPD marker OPD-209n. These results revealed the immense poten- tial of peroxidase isozyme patterns and RAPD as genetic markers for sex identification in H. rhamnoides L.

Tissue Expression and Stock Variation of Isozymes of Stone Flounder(Kareius bicoloratus)

Tissue expression and stock variation of isozymes of stone flounder （Kareius bicoloratus） were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The results indicated that all the isozymes examined were obviously tissue-specific. The expressions of SOD＊, GDH＊, G3PDH-2＊ and ADH-2＊ were detected only in liver, SDH-1＊, MDH-1＊ and ADH-1＊ only in muscle, and LDH-B＊ and LDH-C＊ only in eyes. In comparison, MDH-2＊, GPI-3＊ and SDH-2＊ were detected in all tissues examined. Other loci examined were detected in a variety of tissues. Muscle and liver were selected to detect the isozyme variation of the two geographic stocks of Qingdao and Weihai, Shandong Province, China. The percentages of polymorphic loci （P0.99） were 29.17% and 25.00%, the observed heterozygosities （H0） were 0.028 ±0.014 and 0.040 ± 0.019, and the expected heterozygosities （He） were 0.039±0.017 and 0.052±0.022 in Qingdao and Wethai stock, respectively. The coefficient of gene differentiation （Fst） and genetic distance （D） between the two stocks was 0.012 and 0.0011, respectively, indicating that the genetic differentiation is low between them. Compared with other species of Pleuronectiformes, both the percentage of polymorphic loci and the mean heterozygosity ofK. Bicoloratus were at a middle level.

Effect of Coconut Milk and Rare Earth on Proliferation, Rootage and Isozymes of Dendrobium in Tissue Culture

In tissue culture, effects of coconut milk, rare earth and medium on proliferation, rootage and isozymes of Dendrobium were studied. The result indicated that coconut milk and RE could promote proliferation and rootage, with their combined treatment the most effective. In proliferation culture, coconut milk enhanced peroxidase (POD) and esterase(EST) activity and types, RE decreased POD, but increased EST activity and types and combined treatment did not show the same influence on POD and EST too. In rootage culture, adding coconut milk decreased POD activity, but improved EST activity in 1/2MS medium and declined EST activity, expression type in WPM medium. when using RE, POD and EST activity both reduced, types of the former decreased, types of the latter changed slightly with the basic medium. And combined treatment would decreased POD activity and types.

BIOCHEMICAL GENETIC STUDIES ON CUTTLEFISH SEPIELLA MAINDRONI (CEPHALOPODA: SEPIIDAE)──ACTIVE LOCI SCREENING OF ISOZYME

Screening of 46 putative enzyme coding loci and 4 different kinds of tissues of Sepiella maindroni de Rochebrone, 1884 for enzymatic activities using starch gel electrophoretic technique proved that the 21 enzymes such as AAT, AK, ALP, AP, CK, DIA, ES, FBP, G3PDH, GPI, GRS, IDH, LDH, MDH, MEP, MPI, NP, PGDH, PGM, SOD and XO *, were active to Sepiella maindroni after being stained. The tissue exhibiting stable and clear bands was also determined. Among tissues tested, mantle muscle tissue was the best for electrophoretic survey of isozymes. Buccal bulb muscle, eye and liver were fairly good for some special enzymes, such as DIA, ES, MPI, NP, etc.