Isolation and Characterization of a Class I SLG Gene from Chinese Cabbage (Brassica campestris)

In Brassica species, self-incompatibility in the recognition reaction between self and non-self pollens is determined by two genes, SLG and SRK, at the S locus. We have cloned and characterized a genomic DNA fragment containing a complete open reading frame of the SLG gene from Chinese cabbage. The genomic clone, named BcSLG2, was found to possess the region that shares a homology of 77% in amino acid identity with the SLG46 gene of Brassica campestris. Northern blot analysis revealed that the BcSLG2 gene expression is restricted to the pistil of Chinese cabbage flower. In situ hybridization showed that in the pistil, the gene is expressed predominantly in the stigmatic tissue. Much lower expression in the tapetum was also detectable at an immature stage of the flower development. Southern blot hybridization with the BcSLG2 DNA probe showed polymorphism in the SLG gene organization of the Chinese cabbage plants. These results will provide valuable information in understanding the S gene complex of the Chinese cabbage plants.     

Polymorphism of the S -locus glycoprotein gene ( SLG ) and the S -locus related gene ( SLR1 ) in Raphanus sativus L. and self-incompatible ornamental plants in the Brassicaceae

The S-locus glycoprotein gene, SLG, which participates in the pollen-stigma interaction of self-incompatibility, and its unlinked homologue, SLR1, were analyzed in Raphanus sativus and three self-incompatible ornamental plants in the Brassicaceae. Among twenty-nine inbred lines of R. sativus, eighteen S haplotypes were identified on the basis of DNA polymorphisms detected by genomic Southern analysis using Brassica SLG probes. DNA fragments of SLG alleles specifically amplified from eight S haplotypes by PCR with class I SLG-specific primers showed different profiles following polyacrylamide gel electrophoresis, after digestion with a restriction endonuclease. The nucleotide sequences of the DNA fragments of these eight R. sativus SLG alleles were determined. Degrees of similarity of the nucleotide sequences to a Brassica SLG (S  ⁶ SLG) ranged from 85.6% to 91.9%. Amino acid sequences deduced from these had the twelve conserved cysteine residues and the three hypervariable regions characteristic of Brassica SLGs. Phylogenetic analysis of the SLG sequences from Raphanus and Brassica revealed that the Raphanus SLGs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs. These results suggest that diversification of the SLG alleles of Raphanus and Brassica occurred before differentiation of these genera. Although SLR1 sequences from Orychophragmus violaceus were shown to be relatively closely related to Brassica and Raphanus SLR1 sequences, DNA fragments that are highly homologous to the Brassica SLG were not detected in this species. Two other ornamental plants in the Brassicaceae, which are related more distantly to Brassica than Orychophragmus, also lacked sequences highly homologous to Brassica SLG genes. The evolution of self-incompatibility in the Brassicaceae is discussed. Received: 9 October 1997 / Accepted: 27 January 1998     

Morphological and molecular characterization of somatic hybrid plants between Lycopersicon esculentum and Solanum nigrum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. var. cerasiforme) and of an atrazine-resistant biotype of black nightshade, (Solanum nigrum L.), were fused by using polyethylene glycol/dimethyl sulfoxide (PEG/DMSO) solution and three somatic hybrid plants, each derived from a separate callus, were recovered. A twostep selection system was used: (1) protoplast culture medium (modified 8E) in which only tomato protoplasts formed calluses; and (2) regeneration medium (MS2Z) on which only S. nigrum calluses produced shoots. These selective steps were augmented by early isozyme analysis of putative hybrid shoots still in vitro. Phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT) mapped to five loci on four chromosomes in tomato confirmed the hybrid nature of the nuclei of regenerated shoots. The somatic hybrid plants had simple leaves, and intermediate flower and bud morphology, but anthesis was reduced to 5% due to premature bud abscission and the pollen grains were non-viable. Southern DNA blot hybridization using a pea 45 S ribosomal RNA gene probe reconfirmed the hybrid nature of the nuclear genome of the three plants. A ³²P-labeled probe of Oenothera chloroplast DNA (cpDNA) hybridized to cpDNA restricted with EcoRI or EcoRV indicated the presence of the tomato cpDNA pattern in all three hybrids. Likewise, the plants were all found to be atrazine sensitive. Analysis with two mitochondrial (mt)DNA-specific probes, maize cytochrome oxidase subunit II and PmtSylSa8 from Nicotiana sylvestris, showed that, in addition to typical mitochondrial rearrangements, specific bands of both parents were present or missing in each somatic hybrid plant.Michigan Agricultural Experiment Station Journal Article No. 12433     

NTGLO: a tobacco homologue of the GLOBOSA floral homeotic gene of Antirrhinum majus: cDNA sequence and expression pattern

We report the cloning and DNA sequence of a cDNA from Nicotiana tabacum, NTGLO, as well as the pattern of expression of the NTGLO gene in wild-type tobacco plants. The NTGLO cDNA encodes a protein of 209 amino acids, which shows 73% identity with the GLO protein encoded by the GLO gene of Antirrhinum majus, a homeotic gene involved in the genetic control of flower development. Northern blot analysis shows that the NTGLO gene is expressed mainly in floral organs and, within the flower, expression is restricted to petals and stamens. The NTGLO gene most probably represents a true homologue of the GLO gene because: i) the MADS boxes, of the two genes are highly homologous (56 out of 58 amino acids are identical): ii) at the carboxyterminal a block of 19 amino acids is perfectly conserved between the NTGLO and GLO proteins and iii) their expression patterns in floral organs are identical.     

PCR-RFLP of S locus for identification of breeding lines in cruciferous vegetables

A simple method of detecting polymorphism of S locus glycoprotein gene, SLG, in Chinese cabbage and cabbage was developed, and used for identification of breeding lines. DNA was amplified by the polymerase chain reaction (PCR) with a pair of primers having S ₆ SLG sequences from inbred lines, and digested with restriction endonucleases which recognize tetranucleotide sequences. The cleaved DNA fragments were size-fractionated by polyacrylamide gel electrophoresis and detected by silver staining. PCR with S ₆ SLG primers amplified a fragment of ca. 1.3kb in more than half of the inbred lines tested. After digestion, polyacrylamide gel electrophoresis revealed polymorphism between the amplified 1.3kb DNA fragments. These polymorphic bands were detected by Southern hybridization using a probe of S ₆ SLG cDNA, suggesting that the amplified DNA was SLG. Primers having the SLG sequences of S ₂ , a representative of recessive S alleles, were used for amplification of SLG in the lines which did not give the 1.3kb DNA fragment by the PCR with S ₆ SLG primers. Polymorphism of amplified DNA was found in these lines. However these primers also appeared to amplify an invariant SLR-2 sequence of 1.3kb in addition to the polymorphic S ₂ SLG related sequences. Although the used primer sequences still need improvement for the analysis of recessive S alleles, PCR-RFLP of SLG was considered to be useful for identification of breeding lines as well as for S allele identification in cruciferous vegetables. F₁ hybrids exhibited the sum of the bands of both parents, and, therefore, this method is expected to be used for a purity test of F₁ seeds.     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica. Received: 15 April 1997 / Accepted: 13 June 1997     

StP5CS基因转化结球甘蓝的研究

为研究StP5CS基因在结球甘蓝中的耐盐作用,以结球甘蓝下胚轴为外植体,采用农杆菌介导法将耐盐基因StP5CS和抗除草剂Bar基因导入结球甘蓝基因组中,在双丙氨膦的筛选下扩繁、生根,共获得了36株抗性植株。PCR扩增和Southern印迹杂交检测表明:目的基因StP5CS和Bar基因已经成功导入结球甘蓝基因组中。RT-PCR检测表明:StP5CS基因在转录水平也有表达。转基因植株耐盐试验结果显示:高浓度盐处理(400mmol/L NaCl)下,对照植株整株枯死,而转基因植株仍能正常生长;转基因植株的SOD活性、脯氨酸含量和相对膜透性均随盐浓度的升高呈上升趋势,均在400mmol/L NaCl处理下达到最大。结果表明转基因植株对高盐环境有一定的耐受性。     

油菜糖基转移酶BnIRX14基因家族鉴定及遗传转化

糖基转移酶在植物抗逆和发育调控中发挥着重要作用，为发掘糖基转移酶BnIRX14基因家族成员，解析在甘蓝型油菜中的生物学功能，该研究利用前期在甘蓝型油菜中克隆到的BnIRX14基因，采用序列比对和遗传转化的方法，进行BnIRX14基因家族成员鉴定和功能验证，以探讨BnIRX14基因家族在油菜发育中调控机理，为油菜杂交育种和抗逆育种提供理论依据。结果表明：(1)经基因组数据库比对分析在甘蓝型油菜中成功鉴定到3个糖基转移酶不同亚家族的11个BnIRX14家族成员，它们均具有糖基转移酶GT43家族成员结构域特征，其中有8个基因分别被定位在6条不同染色体上，3个亚家族在基因结构和保守元件中具有较大特异性。(2)利用农杆菌介导转化法，获得BnIRX14基因RNA干扰转基因油菜株系20株，经PCR检测，确定5株阳性转化体。(3)表型鉴定发现，有2株阳性转化株的花柱头至花柱中央为一孔状空腔，子房较野生型明显膨大，且柱头表面授粉后不能结实，表现雌性不育；其他3个阳性株花器结构发育正常，但植株茎、枝表皮有液体渗出，呈露珠状粘附在茎、枝表面。(4)实时荧光定量PCR分析显示，转BnIRX14基因油菜阳性植株...     

Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea

Summary In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S ₂ and S ₆. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S ₆ genotype and 350 kb in the S ₂ genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.     

Visualization of a self-incompatibility gene in Brassica campestris L. by multicolor FISH

 The physical localization of the S-glycoprotein (SLG) locus in the chromosome of Brassica campestris L. ‘pekinensis’ cv ‘Kukai’ was visualized by multi-color fluorescent in situ hybridization (McFISH). ‘Kukai’, which is an F₁ hybrid between two parental lines, T-17 and T-18, has two SLG genes from both T-17 and T-18. In this study, a 1.3-kb DNA fragment was amplified from the genomic DNA of T-17 by PCR using a set of primers specific to the class-I SLG. From the genomic DNA of T-18, no DNA fragment was amplified using these primers. In the genomic Southern hybridization, a cloned PCR product hybridized with the genomic DNA of T-17 or F₁ but not with that of T-18. The PCR product had a sequence homology of approximately, 85% to another class-I SLG gene, SLG-9. Therefore, the PCR product from T-17 was named SLG-17, as it is thought to be a member of the class-I SLG. Using SLG-17 as the probe, FISH was carried out to visualize the position of the SLG locus. McFISH was also carried out simultaneously using the SLG-17 and SLG-9 genes as probes. The SLG-17 gene was detected as a doublet signal at the interstitial region close to the end of a small chromosome, with the signal site being identical to that of SLG-9. Therefore, it is concluded that the SLG-17 gene is localized at the interstitial region close to the end of the chromosome derived from T-17 in Brassica campestris L. ‘pekinensis’ cv ‘Kukai’. Received: 18 September 1997 / Accepted: 6 October 1997     

Characterization of ethylene effects on sex determination in cucumber plants

Sex differentiation in cucumber plants (Cucumis sativus L.) appears to be determined by the selective arrest of the stamen or pistil primordia. We investigated the influence of an ethylene-releasing agent (ethephon) or an inhibitor of ethylene biosynthesis (aminoethoxyvinyl glycine) on sex differentiation in different developmental stages of flower buds. These treatments influence sex determination only at the stamen primordia differentiation stage in both monoecious and gynoecious cucumbers. To clarify the relationships between the ethylene-producing tissues and the ethylene-perceiving tissues in inducing female flowers in the cucumber, we examined the localization of mRNA accumulation of both the ACC synthase gene (CS-ACS2) and the ethylene-receptor-related genes (CS-ETR1, CS-ETR2, and CS-ERS) in flower buds by in situ hybridization analysis. CS-ACS2 mRNA was detected in the pistil primordia of gynoecious cucumbers, whereas it was located in the tissues just below the pistil primordia and at the adaxial side of the petals in monoecious cucumbers. In flower buds of andromonoecious cucumbers, only CS-ETR1 mRNA was detected, and was located in the pistil primordia. The localization of the mRNAs of the three ethylene-receptor-related genes in the flower buds of monoecious and gynoecious cucumbers overlap but are not identical. We discuss the relationship between the mRNA accumulation patterns and sex expression in cucumber plants.     

铁皮石斛DoSMT2基因的功能分析

为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。     

Does the genome of Corylus avellana L. contain sequences homologous to the self-incompatibility gene of Brassica?

Self-incompatibility is a genetic mechanism enforcing cross-pollination in plants. Hazelnut (Corylus avellana L.) expresses the sporophytic type of self-incompatibility, for which the molecular genetic basis is characterized only in Brassica. The hypothesis that the hazelnut genome contains homologs of Brassica self-incompatibility genes was tested. The S-locus glycoprotein gene (SLG) and the kinase-encoding domain of the S-receptor kinase (SRK) gene of B. oleracea L. were used to probe blots of genomic DNA from six genotypes of hazelnut. Weak hybridization with the SLG probe was detected for all hazelnut genotypes tested; however, no hybridization was detected with PCR-generated probes corresponding to two conserved regions of the SLG gene. One of these PCR probes included the region of SLG encoding the 11 invariant cysteine residues that are an important structural feature of all S-family genes. The present evidence suggests that hazelnut DNA hybridizing to SLG differs significantly from the Brassica gene, and that the S-genes cloned from Brassica will not be useful for exploring self-incompatibility in hazelnut.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>) inbred line,Kenshin

We developed a transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin, with high tolerance to soft rot disease. Tolerance was conferred by expression of N-acyl-homoserine lactonase (AHL-lactonase) in Chinese cabbage through an efficient Agrobacterium-mediated transformation method. To synthesize and express the AHL-lactonase in Chinese cabbage, the plant was transformed with the aii gene (AHL-lactonase gene from Bacillus sp. GH02) fused to the PinII signal peptide (protease inhibitor II from potato). Five transgenic lines were selected by growth on hygromycin-containing medium (3.7% transformation efficiency). Southern blot analysis showed that the transgene was stably integrated into the genome. Among these five transgenic lines, single copy number integrations were observed in four lines and a double copy number integration was observed in one transgenic line. Northern blot analysis confirmed that pinIISP-aii fusion gene was expressed in all the transgenic lines. Soft rot disease tolerance was evaluated at tissue and seedling stage. Transgenic plants showed a significantly enhanced tolerance (2–3-fold) to soft rot disease compared to wild-type plants. Thus, expression of the fusion gene pinIISP-aii reduces susceptibility to soft rot disease in Chinese cabbage. We conclude that the recombinant AHL-lactonase, encoded by aii, can effectively quench bacterial quorum-sensing and prevent bacterial population density-dependent infections. To the best of our knowledge, the present study is the first to demonstrate the transformation of Chinese cabbage inbred line Kenshin, and the first to describe the effect of the fusion gene pinIISP-aii on enhancement of soft rot disease tolerance.     

北美鹅掌楸CPP转录因子家族LtTCX2基因的克隆与分析

CPP(cystein-richpolycomb-likeproteinor Tesmin/TOS1-like)家族属于成员数目较少的一类转录因子基因家族,含有保守的富含Cystein的CRC结构域,在植物发育进程中,主要参与花发育、细胞分裂、分子进化等。为了探索CPP转录因子家族在北美鹅掌楸花发育中的作用,该文以北美鹅掌楸(Liriodendron tulipifera)为材料,采用RACE技术克隆出1个CPP-like家族基因,命名为LtTCX2,全长2 866 bp。通过NCBI网站在线分析,ORF长2 424 bp,编码了807个氨基酸,含2个保守的TSO1-like CXC结构域,分子量为88 699.25 Da,理论等电点为5.83,不稳定系数为62.38,疏水性平均值为-0.619,预测为亲水性蛋白、非跨膜蛋白、核蛋白,不含信号肽及切割位点。氨基酸比对及系统进化分析结果显示,LtTCX2与其他物种的CPP家族TCX蛋白具有较高的同源性,与亚洲莲(Nelumbo nucifera)的NnTCX2、胡杨(Populus euphratica)的PeTCX2进化关系最近。荧光定量PCR结果显示,LtTCX2基因在叶片中表达量最高,在萼片、花瓣中几乎不表达,表达量由高至低如下:叶片、花芽、雌蕊、雄蕊、茎、根、花瓣、萼片。以上结果说明,LtTCX2属于较古老、保守的一类基因,可为从分子生物学层面研究鹅掌楸属植物系统进化提供一定的理论依据。