Studies on isozymic variation among the South Indian species of Sphaerostephanos

Objective:To explore the identity and phylogenetic relationships among the three medicinally important species of Sphaerostephanos from South India using isozymic profile.Methods:The young fronds were homogenized with 3.5 mL of ice-cold homogenizing buffer in a pre-chilled pestle and mortar.The supernatant was subjected to electrophoresis as described by Anbalagan poly acrylamide gel electrophoresis.Staining solutions for isoperoxidase was prepared as per Smila method for the detection of isoenzymes.Results:A total of six different bands in five different positions with different molecular weight/Rf values and four active zones have been observed in the isoperoxidase enzyme system of Sphaerostephanos.Only one band with MW/Rf 0.399 is common to two different species i.e.Sphaerostephanos arbuscula(S.arbuscula) and Sphaerostephanos unitus(S.unitus).Among the remaining four bands,two bands(R_f.0.23,0.47)are present in Sphaerostephanos subtruncatus(S.subtruncatus) and one distinct band has been observed individually in S.arbuscula(R_f.0.507) and S.unitus(R_f.0.56).Conclusions:The present preliminary molecular study through isozymic analysis shows the identity of all the three species and the present results confirm distinctness of these three species based on macromicromorphology,phytochemistry and cytology.

Study of Application of Isozymic Marker to Distinguish the Multiplasmic Lines of Maize

On the basis of previous work, the study of screening marker bands of maize ( Zea mays L. )isozyme was carried out. 11 multiplasmic lines were used as experimental materials. The different tissues at different development stages were taken for each material. DPAGE system and double vertical gel slabs were used. The experimental results are as follows: (1) POD: A total of 20 isozymic bands. (Fli)Mo17 and (su1)Mo17 possessed the POD1 and POD3, while POD7 was absent. (sh2)MoI7-POD15 and POD16. (bt1)Mo17-POD1, POD11. (Pop)Mo17-POD15. (wx)Mo17 did not possess the POD3, 4, 5, 6, 8, 15, 16. The other lines possessed different POD. (2) EST: A total of 18 isozymic bands. (Fli)Mo17 possessed the EST 2, 17,but EST15 was absent. (su1)Mo17-EST13. (bt1)Mo17-EST6, 12, 14. (Pop)Mo17 possessed the EST12, but EST2, 8, 11, 13, 14 were absent. For the other multiplasmic lines, some possessed this EST, some possessed other EST. (3)According to the above data, some specific isozymic bands can be used as biochemical markers to distinguish these multiplasmic lines from each other.

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.

Antioxidase System of a Rice Mutant with Low Chlorophyll b

[Objective] The mitigative effect of antioxidase system of a rice mutant with low chlorophyll b on photooxidative damage was studied.[Method] A rice mutant with low chlorophyll b and its wild type were taken as experimental materials to comparatively research their peroxide (H2O2) contents, the activity and isozymes of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in chloroplast.[Result] Compared with the wild type, there were many kinds of SOD, POD and CAT isozymes in leaf cells and chloroplast cell of mutant, and the activity of SOD, POD and CAT isozymes in leaf cells and chloroplast cell of mutant was also correspondingly higher. Under intense light condition, the H2O2 content of chloroplast in mutant was less than that in the wild type. [Conclusion] The higher activity of scavenging active oxygen can relieve the photooxidative damage made by excessive light energy of intense light on photosynthetic membrane, which is an important reason for higher photosystem Ⅱ (PS II) stability of this mutant.

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.

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.

Comparison of Some Characteristics Between Phragmites communis and Its Salt Tolerant Variant

R5002_12, a salt tolerant line of Phragmites communis Trin., which was obtained from ethyl methane sulfonate (EMS) treated callus selected under saline stress, was compared with its wild line in respect to their molecular biological, physiological and biochemical characterizations. Five arbitrary primers were screened which showed differences in DNA amplified polymorphism between the variant and its wild line. Some new proteins appeared in the salt tolerant plant under salt stress. Electrophoresis of peroxidase and esterase also showed some differences in isozyme expression between them. The chlorophyll content of the variant was higher than that of the original variety, whether the plants were under salt stress or not.

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.

Genetic diversity and genetic differentiation of natural populations of Pinus kesiya var. langbinanensis

Genetic diversity and genetic differentiation of natural populations of Pinus kesiya var. langbinanensis were examined by means of electrophoresis technique. Analysis of 9 enzyme systems including 16 loci showed that all the three natural popu-lations of the pine were high in genetic diversity but low in inter-population genetic differentiation. The proportion of polymorphic loci is 0.667, with each locus holding 2.13 alleles, averagely. The average expected and observed heterozygosity was 0.288 and 0.197, respectively. The gene differentiation among populations was 0.052, but the mean genetic distance was only 0.015.

Isoenzymes for Four Coreid Species Identification (Hemiptera: Coreidae)

The vertical slab polyacrylamide gel electrophoretic technique is used to examine the esterase isozyme of four species (Hemiptera: Coreidae), the analysis has revealed that esterase zymogram can be divided into four zones. Esterase zymogram of intergeneric species show distinct difference, while some intrageneric common features are discovered. The clustering analysis shows that the taxonomic status of 4 species, and sex variance is bigger than individuals and is smaller than species.

Characteristics of Cellulases from Anoplophora glabripennis Motsch (Coleoptera: Cerambycidae)

The property of major cellulases from the guts of Anoplophora glabripennis larvae have been characterized. The optimal temperatures of both β 1,4 glucosidase (β glucosidase) and endo β 1,4 endoglucanase (endoglucanase, Cx) are 40℃. The β glucosidase was optimally active at pH 4\^8, while the optimal activity of the endoglucanase occurred at pH 4 4 5 6 The endoglucanase was active with a wide range of pH and temperature, the levels of activity from 25℃ to 50℃ were more than 80%, and the activity remained 60% between pH 3 2 and pH 7 2. The endoglucanase exhibited higher thermal stability than β glucosidase. Both enzymes lose their activities by heat treatment at 60℃. Two isozymes of endoglucanase were detected in sodium carboxymethylcellulose polyacrymide gels (CMC gel) by chemical colorization, and purified by elution from the gel slices. The molecular weights of the two isozymes were estimated as 26kD and 39kD respectively. Moreover molecular characteristics of the two isozymes are currently underway.

Mating Genotype Analysis of Ganoderma lucidum Populations

[Objective] The mating genotype was studied and compared with esterase isozyme of Ganoderma lucidum populations between groups in order to clarify their differences in genetic relationship analysis. [Method] OWE-SOJ technique was applied to identify standard mating types and determinate mating genotype between groups of monokaryons isolates from 24 G. lucidum stains. Genetic relationships were analyzed by combined group mating genotype determination with esterase isozyme assay. [Result] All strains of G. lucidum could be divided into 7 large groups of the mating genotype. Four alleles of A factor, four alleles of B factor and one mixed alleles of A factor were found in this study. Distorted segregation ratio among monokaryon mycelia of G. lucidum had been observed in four kinds of mat- ing types to some extent. Twenty-eight different types of enzyme bands were determined in esterase isozyme test, Twenty-four strains of G. lucidum could be divided into 9 large groups through the cluster analysis when the genetic similarity coefficient was 0.73214. Comparing the results of mating genotype analysis and esterase isozyme analysis, it showed great similarity. [Conclusion] Mating genotype analysis could be used as an important supplementary method for strain identification and genetic diversity research.

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..

葡萄(Vitis)不同种叶片中的过氧化物酶同工酶谱及其与抗病性关系的初步研究

本试验测定不同抗黑痘病葡萄种与品种间健康叶片 Px 酶谱变化,未发现以往有人在农作物上报道过的 Px 酶带有规律的增或减的变化。但试验发现除个别种类外,普遍测得感病叶较健叶 Px 同工酶带色泽加深或酶带增多。分析 Px 同工酶对感病有敏感反应的8个不同抗病性葡萄品种健病叶 Px 同工酶谱变化,发现叶片感病后与抗病性有关的变化酶带有 P_(XA)、P_(XB)、P_(XD)带。本文对3种不同抗病性类型(轻感病型、中感病型、高感病型) Px 同工酶的这些酶带变化规律作了初步分析与讨论。

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.

Polymorphism of Kunitz Trypsin Inhibitor Protein in Natural Populations of Wild Soybean in Hebei

Hebei Province is one of the main distribution areas growing wild soybean( Glycine soja ) in China. In this study, 461 seed samples,collected from 18 natural populations in this province, were used to electrophoretically observe the change in forms and their frequencies of the Kunitz trypsin inhibitor protein (KTI)in individual populations and geographical areas. Allelic frequencies accounted for 85% for Tia and 15% for Tib in the total samples. Twelve populations examined were polymorphic at the KTI locus, accounting for over 50% in the populations investigated. Four populations, 22% of all the populations, were found to have natural cross-pollination with varied heterozygote rates of 3% -5.5%, and the average was 1% in the total sampies. Geographically, the mean Tib frequency in the north areas was higher than in the south, and higher in the mountainous area than in the plain areas. The populations in a lake ecological environment (Baiyangdian Lake) were almost monomorphic. No obvious relationship between the frequency and the geographical distance was observed. In addition, we first found a mutation for the absence of the KTI in wild soybean.

Enhancement of Laccase Activity by Marine-derived Deuteromycete Pestalotiopsis sp. J63 with Agricultural Residues and Inducers

Pestalotiopsis sp. J63, producing a high activity of laccase, is a new marine-derived fungus isolated from the oceanic sediment of the East China Sea. Since the marine environment is oligotrophic nutrient, marine derived fungi may use small amount of nutrients to grow and produce laccases. Agricultural residues that are mainly composed of lignin, cellulose and hemicellulose are difficult to be degraded and few microbes can take them as substrates, so they are considered as oligotrophic nutrient and have the potential to be used to produce value added products. In this study, the ability of Pestalotiopsis sp. J63 to use agricultural residues to produce laccases was tested in the submerged fermentation. The combination of 3 g·L 1maltose and 20 g·L 1rice straw was the best carbon sources and 8 g·L 1ammonium sulfate was the best nitrogen source under the condition without inducers. The effects of five inducers, the feeding time and concentration of inducer on laccase production were investigated.Adding 0.09 mmol·L 1phenol after 24 h of incubation led to high laccase activity(5089 U·L 1), while with 0.09mmol·L 1phenol in the medium and wheat bran as the nitrogen source, the laccase activity could reach 5791.7U·L 1. Native-PAGE results showed that two laccase isozymes were present in the cultures. One existed in both induced and non-induced culture filtrates, while the other was only found in the fermentation with the addition of phenol, guaiacol and veratryl alcohol.

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.