Introduction of mitochondrial DNA from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> into <Emphasis Type="Italic">Pleurotus pulmonarius</Emphasis> by interspecific protoplast fusion

Interspecific protoplast fusion between two monokaryotic strains (a methionine auxotrophic and chloramphenicol-resistant Pleurotus ostreatus strain and a wild-type strain of Pleurotus pulmonarius) was carried out to introduce mitochondrial DNA (mtDNA) from P. ostreatus into P. pulmonarius. Because mycelial colonies regenerated on minimum medium containing chloramphenicol only after the treatment of protoplast electrofusion, the regenerants were regarded as protoplast fusants. The fusants isolated from regenerated colonies were uninucleate, and their resistance of chloramphenicol seemed to be a stable trait. The fusants mated with P. pulmonarius but not with P. ostreatus, and showed almost identical random amplified polymorphic DNA (RAPD) patterns to that of the parental P. pulmonarius monokaryon. The sizes of mitochondrial genomes were estimated to be 81.5 kb for P. ostreatus monokaryon, 54.9 kb for P. pulmonarius monokaryon, and 84.5 or 86.0 kb for the fusants. BamHI and EcoRI digest patterns of the fusant mtDNAs were almost the same as the parental P. ostreatus monokaryon. From the above results, the fusants seemed to carry nuclei derived from the monokaryon of P. pulmonarius and mtDNA including the chloramphenicol-resistance gene from the P. ostreatus monokaryon, suggesting that the P. ostreatus mtDNA had been introduced into P. pulmonarius cells by interspecific protoplast fusion. Contribution No. 383 of the Tottori Mycological Institute     

Analysis of the electrophoretic karyotype ofFlammulina velutipes

The karyotype ofFlammulina velutipes (Curt.: Fr.) Sing. was analyzed electrophoretically using contourclamped homogeneous electric field gel electrophoresis and hybridization with DNA probes. The chromosomal DNA from the monokaryon (Fv-4K) and the dikaryon (Fv-4) were resolved into six and eight bands, respectively. The sizes of the chromosomes ranged from 1.9 to 6.0 megabase (Mb) pairs. Each of the separated bands of chromosomal DNA was identified by use of five cloned probes. The number of these chromosomes was estimated to be 6 and 12, respectively; and the size of the entire genome was estimated to be about 20.1 and 38.6Mb, respectively. From a comparison of the hybridization patterns, the existence of allelic chromosomes of different sizes was deduced in the Fv-4 strain.     

杨树亲-子代叶片的过氧化物酶同工酶分析

通过聚丙烯酰胺不连续垂直板凝胶电泳,对杨树6个全同胞家系的亲本及子代叶片的过氧化物酶(POD)同工酶,及其与生长量和杂种优势之间的关系进行了研究。结果表明:供试单株及不同家系在酶谱带数、迁移率、酶的活性强弱等方面存在一定的差异,即使同一家系内亲代与子代之间也存在差异,子代主要表现为双亲互补型酶带。POD同工酶分析可用于杨树亲缘关系的鉴定。子代与母本的相似系数比子代与父本的相似系数高,偏向母性遗传。同时,研究结果还显示过氧化物酶酶带数和酶强度与地径生长量之间均呈显著性正相关,相关系数分别为0.89和0.83;酶带数和酶强度与地径超亲优势之间也呈显著性正相关,相关系数分别为0.88和0.84;而酶带数和酶强度与苗高生长量及苗高超亲优势之间的相关性均不显著。酶带的谱型与生长量和杂种优势之间也没有表现出明显的相关性。因此,仅从过氧化物酶同工酶酶谱的带数、强弱和谱型来判断杨树的生长量和杂种优势是不完全合理的。     

应用同工酶鉴定黑松林内变异类型的遗传特性

黑松(Pinus thunbergii Parl.)原产于日本,引入我国山东烟台沿海地区巳有70多年历史,生长优于当地的赤松(Pinus densiflora Sieb.et Zucc),抗松干蚧的能力高于赤松。解放后,黑松成为营造海防林的主要造林树种。在选材的黑松林内,约有90％以上的个体是黑松,另有不足10％个体的干形、皮色、树皮开裂、叶形、叶色、冬芽大小、颜色……等都不     

Transmission of mitochondrial plasmids in protoplast cell fusion between compatible monokaryons of <Emphasis Type="Italic">Lentinula edodes</Emphasis>

Electrophoretic analysis of the transmission pattern of mitochondrial plasmids in protoplast cell fusion between compatible monokaryons of Lentinula edodes indicates that three of the four plasmids carried in parental monokaryons are effectively transferred and replicated in the protoplast fusants. The two monokaryons, 1158a and 1569a, carried different plasmids that could be distinguished by a single restriction digest. Electrophoresis of intact plasmids and restriction analyses indicate that all but one of the fusants carry three of the four possible plasmids, indicating that transmission of plasmids in protoplast fusions is principally biparental in L. edodes. Thus, heterocytoplasmic cells of L. edodes can be effectively constructed by protoplast cell fusion. In addition, plasmids of the same homology group cannot coexist in the heteroplasmic cells after protoplast cell fusion. Contribution No. 382 of the Tottori Mycological Institute     

Study on isozyme electrophoresis ofMartes zibellina L.

Four isozymes, such as Malate dehydrogenase (MDH), Alchol dehydrogenase (ADH), Peroxidae (POD) and Esterase (EST) in six tissues (heart, liver, kidney, muscle, eye, gonad) ofMartes zibellina L., were analyzed by means of vertical polyacrylamide gel electrophoresis (PAGE). The results indicated that the zymograms of these four isozymes in different tissues were different from each other, i.e. there existed apparent tissue-specificitity in these isozymes inMartes zibellina L.. Characteristic enzyme band was found both in POD zymogram and in EST zymogram. Moreover, the characteristic enzyme band in POD isozyme would be of some value to sexual identification ofMartes zibellina L. (Responsible Editor: Zhu Hong)     

Variation of fruiting body productivity in protoplast fusants between compatible monokaryons ofLentinula edodes

Mycelial protoplasts of two compatible monokaryons ofLentinula edodes were prepared, and electro-fusion was undertaken. Sixteen dikaryons isolated after the electrofusion were tested for their fruiting abilities in comparison to the two dikaryons obtained by reciprocal mycelial mating. There were significant differences in fruiting body yield among the fusants, and some of them produced more fruiting bodies than did normally mated dikaryons. Although these dikaryons were divided into five genetically different groups by isozyme analyses and a cluster analysis, we could not find clear differences among the groups in terms of fruiting body yield, mycelial growth rate, or degree of wood decay. It appeared that fruiting body yield correlated positively with the mycelial growth rate but negatively with the degree of wood decay.     

An assessment of heterozygosity and fitness in Chir pine (<Emphasis Type="Italic">Pinus roxburghii</Emphasis> Sarg.) using isozymes

A correlation between heterozygosity of genotypes and survival (fitness) was studied in eight natural populations of Pinus roxburghii through isozyme analysis using horizontal starch gel electrophoresis. Under each population, 20 mother trees of different ages (30 to more than 100 years) were selected which were at least 50 m apart. From each tree, eight seeds were assayed for eleven enzyme systems viz., Aconitase, Aspertate aminotransferase, Glutamate dehydrogenase, Isocitrate dehydrogenase, Leucine-amino peptidase, Malate dehydrogenase, Menadione reductase, Phosphoglucose isomerase, Phosphoglucomutase, 6-Phosphogluconate dehydrogenase and Shikimic acid dehdrogenase which were found to be encoded by 18 polymorphic loci. Genetic constitution of the mother trees was determined by analysing endosperms and embryos separately. The number of alleles in the progeny (embryos) and the mother trees varied from 38 to 42 and 34 to 37, respectively, in all the populations. Distribution of degree of heterozygosity and fixation index for the progeny and the mother trees were calculated. The mother trees showed a substantial shift towards higher degree of heterozygosity as compared to the progeny. Fixation index values were significantly higher and negative for the mother trees as compared to the progeny, which revealed that heterozygosity was positively correlated with survival in P. roxburghii. Application: Heterozygous geneotypes in tree species are shown to have better survival over homozygotes under diverse environmental conditions. Every year, millions of Pinus roxburghii saplings are planted in India. However, the fact that survival is very low might possibly be due to inbred/homozygous plants that suffer from fitness disadvantage. Our study indicates that heterozygotes have a better chance of survival under environmental conditions. The use of heterozygotes should lead to effective establishments of Pinus roxburghii plantations.     

Cladistic relationships among the Pleurotus ostreatus complex, the Pleurotus pulmonarius complex, and Pleurotus eryngii based on the mitochondrial small subunit ribosomal DNA sequence analysis

The cladistic analysis of the V4 domain sequences, performed by UPGMA, the neighbor-joining, and parsimony methods, revealed that the 19 Pleurotus strains tested in this study evolved along three lineages, each corresponding to a separate biological species: the Pleurotus ostreatus complex, the Pleurotus pulmonarius complex, and Pleurotus eryngii. Moreover, the cladistic positions of the 3 biological species show that the P. ostreatus complex and P. eryngii were derived from a common ancestor at a later stage of evolution, and that the common ancestor had diverged from the lineage of the P. pulmonarius complex during an earlier evolutionary event. The sequences of the 5 portion of the mt SSU rDNA among the strains of the P. ostreatus complex had 99.2%–99.6% homology. All test strains in the P. pulmonarius complex had completely identical sequences. The homology of the strain sequences between the P. ostreatus complex and the P. pulmonarius complex ranged from 96.0% to 96.3%. The sequence of the strain of P. eryngii showed 97.8%–98.3% and 96.5% homologies with those of the strains in the P. ostreatus and the P. pulmonarius complexes, respectively.     

Assessment of intrapathovar variability of Xanthomonas axonopodis pv. commiphorae through phenotypic and molecular markers

Gummosis of guggal (Commiphora wightii) caused by Xanthomonas axonopodis pv. commiphorae (Xac) is one of the major reasons for drastic reduction in guggal population under natural plant stand. The pathogen spreads mainly through human activities (tapping). We isolated 43 Xac strains from 14 locations representing four different regions spread over three districts of Gujarat state, India. A polyphasic approach was followed to characterize these strains and to measure phenotypic and genetic variations among them. All strains were identical in colony morphology and were virulent on guggal. Some of the strains showed differential reactions towards certain biochemical tests viz., acid production from carbon sources. Whole cell protein profiles were apparently uniform for all strains with similarity coefficients ranging between 0.75 and 1.00. Clustering based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis banding patterns showed heterogeneity among the isolates, but strains originating from same zone had similar protein profiles. Inter simple sequence repeats (ISSR)‐based and repetitive elements (rep)‐based PCR analyses revealed genetic heterogeneity among the strains. Both these methods were equally effective in deciphering variability among the strains and indicated similar types of variability with highly significant correlation (r = 0.91) on the similarity matrices. The variation matrix analysis on combined ISSR‐ and rep‐PCR data suggested 66.1% variability among Xac strains. The present study established that Xac strains from different geographical locations had profound genetic heterogeneity.     

Electrophoretic protein profiles of strains of Ceratocystis fimbriata f. sp. platani

Soluble proteins extracted from the mycelium of strains of Ceratocystis fimbriata (Ell. et Halst) Davidson f. sp. platani Walter were separated on native and SDS-polyacrylamide gradient gel electrophoresis. The protein patterns were compared and the electrophoretic profiles of all the strains examined showed striking intraspecific similarities. The use or protein banding patterns as a diagnostic criterion to distinguish strains of C. fimbriata is discussed.     

Enzyme variation among some southwest Turkey populations of the two-spotted spider mite, Tetranychus urticae Koch

Ten different two-spotted spider mite (Tetranychus urticae Koch) populations were collected from greenhouses in two geographically different regions of Turkey, Antalya and Isparta. Individual spider mites were homogenized in microplates using multi-homogenizer, and the genetic variations in the esterase of these populations were studied by nondenaturing polyacrylamide gel electrophoresis. The results showed that esterase enzymes were polymorphic in all populations in both regions. However more intrapopulation and interpopulation variations were observed in the population from Antalya region.     

“金丝油桐”与贵桐2号的过氧化氢酶同工酶谱

为了从生化水平上鉴别"金丝油桐"的品种,利用SDS-聚丙烯酰胺凝胶电泳技术,分析了湖北省来凤县"金丝油桐"与贵桐2号叶的过氧化氢酶同工酶谱,结果显示:"金丝油桐"有11条酶带,其中a、b、f、i、l这5条酶带颜色较深,很清晰,而c、e、g、h、j、k这6条酶带颜色较浅;贵桐2号共有13条酶带,其中a、b、f、i这4条酶带颜色很深,而c、d、e、g、h、j、k、l、m这9条酶带颜色较浅;贵桐2号的酶带比"金丝油桐"多出d、m这2条;"金丝油桐"11条酶带的Rf值依次为0.20、0.36、0.37、0.43、0.46、0.50、0.52、0.59、0.68、0.76、0.90;贵桐2号13条酶带的Rf值依次为0.20、0.36、0.37、0.39、0.43、0.46、0.51、0.52、0.60、0.69、0.77、0.88、0.95。分析结果表明:"金丝油桐"与贵桐2号这2个品种间存在着亲属关系,但其差别较大。     

Duplication of locus coding of malate dehydrogenase in Populus tomentosa Carr.

Horizontal starch-gel electrophoresis was used to study crude enzyme extraction from young leaves of 234 clones of Populus tomentosa Carr. selected from nine provenances in North China. Ten enzyme systems were resolved. One hundred and fifty-six clones showing unusual allozyme band patterns at locus Mdh-1 were found. Three allozyme bands at locus Mdh-1 were 9:6:1 in concentration. Further studies on the electrophoretic patterns of ground mixed pollen extraction of 30 male clones selected at random from the 156 clones were conducted and it was found that allozyme bands at locus Mdh-1 were composed of two dark-stained bands and a weak band. Only one group of the malate dehydrogenase (MDH) zymogram composed of two bands was obtained from the electrophoretic segregation of pollen leachate of the same clones. A comparison of the electrophoretic patterns one another suggested that the locus Mdh-1 coding malate dehydrogenase in diploid species of P. tomentosa was duplicated. The duplicate gene locus possessed three same alleles and was located in mitochondria. The locus duplication of alleles coding malate dehydrogenase in P. tomentosa was discovered and reported for the first time. [Supported by the “Tenth Five-year Plan” National Key Project in Science and Technology (Grant No. 2002BA515B0303) and the National “863” Project (Grant No. 2002AA241071)]     

Relationship between witches’ broom protein and dynamic of some amino acids inPaulownia tree leaves

This paper dealt with the SDS polyacrymide gel electrophoresis of proteins and analysis of the amino acids in the diseased and healthy leaves with the same strain respectively, which were at the same side and height, ofPaulownia catalpifolia, P. elongata, P. albiphloea andP. kauakamii respectively. The results indicated that the leaves of 4 species ofPaulownia trees with witches’ broom had one protein band, of which molecular weight was 12 kD, which did not appear in the healthy leaves free of phytoplasmas. Moreover, the protein quantity in the affected leaves was more than that in the healthy leaves free of phytoplasmas; At the same time, there was significant difference on the amino acids between the healthy and diseased leaves ofP. catalpifolia andP. kawakamii. The amount of cystine in the affected leaves was higher than that in the healthy leaves, but the change of amount of phenylalanine in the affected and healthy leaves was contrary. These changes of proteins and amino acids in the leaves might be related to the witches’ broom of theParlounia trees. (Responsible Editor: Dai Fangtian)     

Electrophoretic pattern of proteins isolated from immatured and matured stages of 10 candidate plus trees of versatile oleaginous legume, <Emphasis Type="Italic">Pongamia pinnata</Emphasis> (L.) Pierre

Pongamia pinnata an oil yielding leguminous tree is an economically important plant due to its multifunctional characteristics. The objective of the present study was to investigate the electrophoretic spectra sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of total soluble seed proteins under reduced conditions (5 mM β-mercaptoethanol) in immatured [135 days after flowering (DAF)] and matured stages (350 DAF) for 10 tagged candidate plus trees (CPTs) of P. pinnata from North Guwahati. SDS-PAGE analysis revealed presence of ~15–20 unique polypeptide fragments of molecular size ranging from 14 to 150 kDa in both stages of seed development but no variations was observed at a particular stage among 10 CPTs. However, polypeptide banding pattern of immatured and fully ripened matured seeds showed differences in expression pattern of three main polypeptide bands (MW 50, 18 and 14 kDa) for all the 10 CPTs studied. Globulins may be considered to be the main seed storage proteins in Pongamia that includes legumins and vicilins.     

Endochitinase activity in the apoplastic fluid of Phellinus weirii‐infected Douglas‐fir and its association with over wintering and antifreeze activity

Extracellular proteins were extracted from Phellinus weirii infected Douglas‐fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase‐like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two‐dimensional gel electrophoresis (2‐D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27–30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii‐infected winter Douglas‐fir needles showed anti‐freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response.     

Production of a polyclonal antibody to Phellinus pini and examination of its potential use in diagnostic assays

In order to differentiate among Phellinus pini, Inonotus tomentosus and Inonotus circinatus a polyclonal antibody was raised to a N-terminal part of 25-kDa P. pini-specific protein. The specificity of the polyclonal antibody produced against a synthetic N-terminal peptide of this protein was investigated for diagnostic purposes using Western immunoblot, indirect enzyme-linked immunosorbent assay (ELISA), and inhibition ELISA techniques. The N-terminal synthetic peptide, used as the immunogen, was found to be more than 80% pure by reverse-phase high-performance liquid chromatography. Following immunization, antisera were collected at three different time intervals. The antibody molecules were purified from the crude antisera using immunoaffinity gel chromatography. Following one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis, Western immunoblot analysis showed that the P. pini I polyclonal-antibody detected the immunogen, the 25-kDa protein, in all but one of the P. pini isolates examined, but in none of the isolates of the nontarget species I. tomentosus and I. circinatus. Nevertheless, cross-reactivity was a problem because the P. pini I polyclonal-antibody also recognized bands at other molecular weights in nearly all of the isolates of the other species tested. With the indirect ELISA the P. pini isolates tended to have higher affinity for the polyclonal antibody than the nontarget species, but some cross-reactivity did occur. Inhibition ELISAs, performed over a range of soluble antigen concentrations (1.56–400 ng/100 μl), failed to show a clear distinction between P. pini and the two Inonotus spp. The low level of cross-reactivity observed for I. tomentosus isolate 52 (9%) was also apparent in the indirect ELISA analysis. All three assays indicated that P. pini isolate 41 was the most antigenic. Despite cross-reactivity, the antibody is useful in Western immunoblots for the diagnosis of most P. pini isolates.     

Lectin from the trunk of Sophora japonica

Summary Lectin was purified from the bark of Sophora japonica trunk by a simple method, affinity chromatography on acid-treated agarose; the yield was about 27% of the total sap proteins. Its molecular weight was 135,000±5,000 shown by using gel filtration, similar to that of the seed lectin of this species. When analyzed by using polyacrylamide gel electrophoresis (PAGE) under acidic and basic conditions, the lectin showed multiple bands. These patterns were different from those obtained with the seed lectin of the same tree. The trunk lectin had a broader specificity for the blood types than the seed lectin. These observations demonstrate that the trunk and the seed lectins from the same tree are different molecular species.Authors are grateful to D. Med. Sci. Kazuhiko Ito and M. D. Hisahiro Yoshida, University Hospital of Kyoto Univ., for kindly providing us with human erythrocytes several times