发根农杆菌K599对大豆、黄瓜和凤仙花活体感染生根的研究

利用野生型的发根农杆菌K599对属于不同科属的大豆、黄瓜和凤仙花进行活体感染实验,结果表明,发根农杆菌K599可使切割后的大豆、黄瓜和凤仙花子叶形成不定根,生根频率分别为100%、65%和91%。此外,发根农杆菌K599还可以使未进行创伤切割的黄瓜腋芽处形成不定根,生根频率为10%。根据发根农杆菌所具有的rolC基因序列,设计PCR引物,对再生的毛状根DNA进行PCR扩增,验证了再生的毛状根中含有发根农杆菌T-DNA序列。本研究获得的转基因根为进一步开展大豆、黄瓜的根结线虫病理以及凤仙花的矮化育种研究提供了前提材料。     

Isolation and characterization of cDNA clones for plant cyclins.

We have isolated and sequenced a carrot cDNA and two soybean cDNAs encoding mitotic cyclin homologs. The soybean clones were derived from nearly identical cognate genes. The carrot cyclin and soybean cyclins were slightly more similar to A-type and B-type cyclins thus far defined, respectively. However, they had divergent amino acid sequences in the portion that is most highly conserved in known cyclins and we could not easily include them in either of the phylogenetic types. Since the homology between carrot and soybean cyclins was low, each of them might define a novel and distinct type. The mRNA of carrot cyclin, 1.5 kb in length, was expressed concomitant with somatic embryogenesis of cultured cells. Expression of soybean cyclin mRNAs, 1.6 kb in length, was localized in proliferating parts of seedlings. As in the case of cyclin genes of marine invertebrates, microinjection of a synthetic mRNA for the soybean cyclin induced the maturation of Xenopus oocytes. Other cyclin genes may be present because, on Southern blot analysis of soybean genomic DNA, the isolated soybean cDNA probe hybridized with additional genes under low stringency.     

Constancy of rDNA Content during Dedifferentiation of Carrot and Jerusalem Artichoke Explants

When carrot explants were cultured with phytohormones, DNA synthesistook place synchronously in the explants and a satellite DNAwith a heavier density in CsCl than the bulk DNA replicatedin the earliest phase of the first replication period. The earlyreplicating carrot satellite consisted of a component havingan identical density to carrot rDNA and another component havinga density between the p-value of carrot rDNA and that of thebulk DNA. DNA-rRNA hybridization was used to explore the possibilitythat this early replication of the satellites leads to amplificationof rDNA in the explant cells, in which massive ribosome synthesisis known to occur. The results showed that there was neitheramplification nor underreplication of rRNA genes during callusformation and its growth. Experiments with explants of Jerusalem artichoke tuber, whichare well known as a synchronous replication system, showed thata component slightly heavier than the bulk DNA was synthesizedat the early phases of the first replication period. However,the density of this early replicating satellite differed fromthat of artichoke rDNA. DNA-rRNA hybridization experiments againshowed no gross changes of rDNA content during dedifferentiationof this plant system. (Received September 30, 1981; Accepted January 5, 1982)     

Repeated sequences in the DNA of the eukaryotic genome

Long DNA molecules from a cucumber satellite, the cucumber main band, mung bean, and Chinese hamster ovary (CHO) were digested with mung bean nuclease I, which was used as a probe for high AT regions. The digests were viewed under the electron microscope, and the distribution of sizes for the fragments of nuclease-treated plant DNA showed that the main band cucumber and the mung bean have regions along their genomes spaced at approximately 0.3 to 0.4 μ that are sensitive to the nuclease. The satellite from the cucumber contains these sites at intervals generally of 0.1 μ or less, whereas CHO DNA has these regions at intervals of 0.05 to 1.40 μ in length. The long DNA from the main band of the cucumber and the CHO were also partially melted in formamide at 37°C to denature preferentially the regions along the DNA molecules that are rich in AT. Measurements of the distances from the center of each loop to the center of the adjacent loops showed that these distances for the main band cucumber DNA tended to occur at approximately every 0.4 μ, whereas the corresponding distances for the Chinese hamster DNA were less regular, occurring every 0.1 to 1.0 μ.     

Pythiogeton zizaniae, a new species causing basal stalk rot of water bamboo in Taiwan

A new species, Pythiogeton zizaniae, was isolated from diseased water bamboo (Zizania latifolia) in central Taiwan. The organism formed a colony with scanty mycelia and mycelial aggregates on rye-water bamboo medium. Special treatments were required for production of sporangia which were terminal, noncaducous and mostly ovoid. Chlamydospores were absent. The fungus was homothallic. Oogonia produced on V-8 water bamboo medium in water were mostly globose to subglobose and each was attached with a club-shaped, monoclinous antheridium by the base of the oogonium stalk. Oospores were plerotic and globose to subglobose. Py. zizaniae caused death of water bamboo suckers but did not infect seedlings of corn, rice, wheat, sorghum, cucumber, tomato, soybean or water spinach. It also did not affect cucumber and tomato fruit, carrot roots or potato tubers.     

Insertion and amplification of a DNA sequence in satellite DNA of Cucumis sativus L. (cucumber)

Summary Another satellite DNA repeat (type IV) in the genome of Cucumis sativus (cucumber) was found and investigated with respect to DNA sequence, methylation, and evolution. This satellite shows a repeat length of 360 bp and a GC-content of 47%. The repeats of type IV are highly conserved among each other. Evidence for CG and CNG methylation is presented. By comparison to the previously described satellites (type I/II and type III) from cucumber, it is evident that this repeat is created by an insertion of a 180 bp DNA sequence similar to type I–III into another DNA sequence (or vice versa), and subsequent amplification forming a new satellite repeat. The different satellites of the type I/II, type III, and the 180 bp insert of type IV show a sequence homology of 60%–70%, indicating that the complex satellite DNA of cucumber is originated from a common progenitor by mutation, additional insertion, and amplification events. Copies of a sequence similar to a part of type IV are present in the genome of the related species Cucumis melo (melon).     

Molecular evolution of two actin genes from carrot

We have isolated and sequenced two full-length cDNA clones encoding actin from carrot. The two carrot clones are almost identical at the nucleotide level, and are quite homologous to each other and to other plant actins at the amino acid level. In those regions where amino acid variation exists between the two genes from carrot, the differences have arisen from very simple changes at the nucleotide level. The most common changes are nucleotide insertion(s) coupled to the deletion of a different nucleotide(s) nearby in the DNA sequence, resulting in the restoration of the proper reading frame for the protein; thus, these changes can be viewed as multiple or coupled frameshift mutations. There are almost no base substitutions between the two carrot genes. In contrast to this, when the carrot actin nucleotide sequences are compared to those of a soybean actin gene or a maize actin gene, many base substitutions are observed (ca. 21.8% and 23.5%), more than half of which are third base changes which do not alter the protein sequence. At the amino acid level, both carrot genes show greater similarity to maize actin than they do to soybean actin, thus reinforcing the idea that plant actin genes diverged from a single common ancestral actin gene prior to the divergence of monocots and dicots.     

Variation in satellite DNA from some higher plants

Pure satellite DNAs were prepared as minor components after centrifugation to equilibrium on CsCl gradients. A single satellite DNA band was isolated from flax (Linum usitatissimum) DNA and two bands were resolved in cucumber (Cucumis sativus) DNA. These apparently homogeneous components of the plant genomes were further analyzed by thermal denaturation and renaturation. The flax satellite DNA appeared homogeneous on thermal denaturation but was shown to contain several components of renaturation. The two cucumber satellite DNAs were different from each other, but both showed at least two components in denaturation and renaturation analyses. Renaturation in the three satellites, particularly in flax, was inaccurate, indicating a considerable degree of sequence divergence. Although each satellite contained quite large amounts of simple repetitious sequences, a residual heterogeneous DNA fraction was always present. It is considered that this was too large a portion of the satellite DNA to be due to organelle or ribosomal DNA in cucumber. The latter possibility is precluded in flax, where the satellite is completely resolved in buoyant density from both organelle and ribosomal DNA.     

Olfactory experience modifies the effect of odour on feeding behaviour in a goal-related manner

Natural food odours elicit different behavioural responses in snails. The tentacle carries an olfactory organ, and it either protracts toward a stimulating carrot odour or retraces in a startle-like fashion away from a cucumber odour. The tentacle retraction to cucumber was still present after the snails were fed cucumber during inter-trial periods. Also, snails without any food experience displayed a longer latency to the first bite of cucumber than of carrot and rejected cucumber more often. After tasting these foods, the latency to carrot was not affected while the latency to and number of rejections of cucumber decreased. These results suggest that initial repulsive features of food odour can be only partially compensated by olfactory learning and feeding experience. In the present study, we demonstrated that an invertebrate can be repulsed or attracted by the same natural odour at the same time and that these behavioural responses are likely aimed at achieving different physiologically relevant goals.     

Early Replication of Highly Repeated DNA Sequences during Dedifferentiation of Carrot

When carrot explants are placed on an agar medium containingphytohormones, a satellite DNA begins to replicate earlier thanthe bulk DNA during the first cell division cycle. The majorityof this early replicating satellite DNA was previously shownto have an identical buoyant density to ribosomal DNA (rDNA)(Hase et al. 1982). Molecular sizes of EcoRI-digests of ³H-labeledDNA were analyzed in the present study by gel electrophoresisfollowed by fluorography. Most of the labeled DNA bands didnot correspond to EcoRI-digests of carrot rDNA. The resultsindicated that the majority of the early replicating satelliteDNA is not rDNA, but probably a type or types of highly repeatedDNA sequences. (Received June 6, 1985; Accepted November 4, 1985)     

不同寄主植物对截形叶螨生长发育及繁殖的影响

在28℃下用叶盘法研究了５种寄主植物对截形叶螨生长发育及繁殖的影响。结果表明,在不同寄主植物上,截形叶螨各螨态历期、单雌产卵量、日均产卵量、产卵期、净增殖率、周限增长率、内禀增长率、世代平均周期和种群加倍时间等均有明显差异; 雌螨完成一代所需时间长短依次为9.3天（黄瓜）、9.3天（菜豆）、9.6天（大豆）、11.0天（茄子）和11.6天（玉米）,产卵期依次为9.9天（茄子）、11.2天（黄瓜）、12.9天（菜豆）、15.8天（玉米）和17.9天（大豆）;单雌产卵量和净增殖率在大豆上最高,分别为115.0和51.7,而在茄子上最低,分别为38.0和11.7。幼、若螨期存活率大小依次为93.6%（大豆）、91.7%（玉米）、89.9%（黄瓜）、84.3%（菜豆）和61.6%（茄子）,存活曲线均为Ⅰ型。大豆是其最嗜食寄主植物,其次是玉米,再次为黄瓜和菜豆,对茄子的嗜食性最差。     

Somatic hybridization in higher plants.

Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two non-allelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco.     

Induced plant responses to pathogen attack. Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy 63).

In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2', 4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.     

Somatic hybridization in higher plants

Summary Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two nonallelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976.     

用衔接头PCR克隆新的胡萝卜Ⅱ型转化酶基因启动子

 为克隆新的胡萝卜 型转化酶基因启动子 ,将胡萝卜基因组 DNA分别用 Pvu 、Eco R 、Dra 和 Sma 酶切 ,酶切片段与一特殊的衔接头连接 .取连接产物作模板 ,以衔接头引物和基因特异引物做 PCR,得到的主要 PCR产物分别为 3.4kb、1 .3kb、0 .4kb和 0 .6kb.将 Eco R -衔接头体系的 PCR产物克隆和测序 ,并将其序列与 Gen Bank中的已知序列进行比较分析 ,发现了一个新的胡萝卜 型转化酶启动子序列 ,它含有类似于 TATA box和 CAAT box的元件 ,在启动子的远上游区域还含有多个 AT富含区 .该启动子的发现对于研究植物中糖代谢具有重要意义 .     

Age‐stage,two‐sex life tables of Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) with a discussion on the problem of applying female age‐specific life tables to insect populations

Abstract Age‐stage, two‐sex life tables of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), reared on cucumber (Cucumis sativus L.), sponge gourd (Luffa cylindrica Roem) and a carrot medium (mashed Daucus carota L. mixed with sucrose and yeast hydrolysate) were constructed under laboratory conditions at 25 ± 1°C, 65%± 0.5% relative humidity, and a photoperiod 12 : 12 h (L : D). The intrinsic rates of increase of B. cucurbitae were 0.144 6, 0.141 2 and 0.068 8 days on cucumber, sponge gourd, and carrot medium, respectively. The highest net reproduction rate was 172 offspring per fly reared on sponge gourd. The mean generation times of B. cucurbitae ranged from 34 days reared on cucumber to 56 days reared on carrot medium. The life history raw data was analyzed using the traditional female age‐specific life table and compared to results obtained using the age‐stage, two‐sex life table. When the age‐specific female life table is applied to an age‐stage‐structured two‐sex population, survival and fecundity curves will be improperly manipulated due to an inability to include variation in preadult development time. We discussed different interpretations of the relationship between the net reproductive rate and the intrinsic rate of increase to clarify possible misunderstanding in the literature.     

Differential replication of satellite and main band DNA during early stages of callus formation in carrot root tissue

DNA synthesis during the early stages of callus formation wasexamined in carrot root slices cultured on an agar medium containing2,4-D. During the first 12 hr of culture, only a low level of3H-thymidine was incorporated into DNA, after which the incorporationrapidly increased and reached a maximum at about 48 hr, thengradually decreased. CsCl density centrifugation of total tissueDNA indicated that a satellite DNA with a buoyant density ofabout 1.712 g/ml replicates in the early phase of the DNA syntheticperiod, then the main band DNA with a buoyant density of 1.695–1.700g/ml starts its replication and continues to be produced throughoutthe synthetic period. The labeled satellite DNA, as well asthe labeled main band DNA, was localized mainly in the subcellularfraction of 1,000 ? g sediments obtained from tissue homogenates.The patterns of cellular localization were not modified by theaddition of Triton X-100 to the initial homogenates. A considerableportion of the labeled satellite DNA was found in nuclease-resistantchromatin subunits after limited digestion of the isolated chromatinwith micrococcal nuclease. (Received August 30, 1979; )     

Divergence, differential methylation and interspersion of melon satellite DNA sequences.

Melon (Cucumis melo) satellite DNA consists of two components, Q and S, each with a buoyant density in CsCl of 1.707 g/ml, but differing by 9 degrees C in "melting" temperature. These physical properties appear to be in contradiction, since both depend on G + C content. In order to resolve this anomaly, base compositions were directly determined for isolated fractions. the low-"melting" component S contains 41.8% G + C, with 6% of C present as 5-methylcytosine, whereas Q DNA contains 54% G + C, with 41% of C methylated. Analyses of restriction site loss agreed well with the direct determinations of methylation and divergence, and indicated some clustering of methylated sites in Q DNA. Analysis of restricted main-band DNA by hydridization with RNA complementary to Q satellite DNA ("Southern transfer") showed satellite Q tandem arrays interspersed in DNA of main-band density. Sequence divergence and extent of methylation did not appear to depend on whether a repeat array was present as satellite or interspersed in main-band DNA. Hydridization in situ indicated considerable heterogeneity in the genomic proportion of the Q-DNA sequences in melon fruit nuclei, implying over- and under-representation consistent with extensive unequal recombination in satellite Q tandem arrays. The cucumber, Cucumis sativus, contains less than 8% as much Q-homologous DNA per genome as the melon, suggesting rapid evolutionary gain or loss of these tandem repeat sequences.     

国家种质库保存大豆和菜豆种质的种传病毒检测

以国家种质中期库提供的300份大豆、100份菜豆种质为材料,分别采用血清学和分子生物学方法,对种传病毒的种类进行了检测。结果表明:在大豆种质中检测出大豆花叶病毒(SMV)、黄瓜花叶病毒(CMV)、苜蓿花叶病毒(AMV)3种病毒,阳性检出率分别为25.33%(76份)、13.67%(41份)和4.67%(14份)。大豆种质中还存在大豆花叶病毒与黄瓜花叶病毒、大豆花叶病毒与苜蓿花叶病毒的复合侵染。在菜豆种质中检测出菜豆普通花叶病毒(BCMV)阳性材料92份,种质带毒率高达92%。这些信息将会对今后采取相关措施提高国家种质库保存的豆类种质的质量提供帮助。     

Construction of a fosmid library of cucumber (Cucumis sativus) and comparative analyses of the eIF4E and eIF(iso)4E regions from cucumber and melon (Cucumis melo)

A fosmid library of cucumber was synthesized as an unrestricted resource for researchers and used for comparative sequence analyses to assess synteny between the cucumber and melon genomes, both members of the genus Cucumis and the two most economically important plants in the family Cucurbitaceae. End sequencing of random fosmids produced over 680 kilobases of cucumber genomic sequence, of which 25% was similar to ribosomal DNAs, 25% to satellite sequences, 20% to coding regions in other plants, 4% to transposable elements, 13% to mitochondrial and chloroplast sequences, and 13% showed no hits to the databases. The relatively high frequencies of ribosomal and satellite DNAs are consistent with previous analyses of cucumber DNA. Cucumber fosmids were selected and sequenced that carried eukaryotic initiation factors (eIF) 4E and iso(4E), genes associated with recessively inherited resistances to potyviruses in a number of plants. Indels near eIF4E and eIF(iso)4E mapped independently of the zym, a recessive locus conditioning resistance to Zucchini yellow mosaic virus, establishing that these candidate genes are not zym. Cucumber sequences were compared with melon BACs carrying eIF4E and eIF(iso)4E and revealed extensive sequence conservation and synteny between cucumber and melon across these two independent genomic regions. This high degree of microsynteny will aid in the cloning of orthologous genes from both species, as well as allow for genomic resources developed for one Cucumis species to be used for analyses in other species. Names are necessary to report factually on available data; however, the US Department of Agriculture (USDA) neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.