Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation method, Arabidopsis transgenic Kan^R plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls, upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any expression in sepals and pistils.     

Phylogenetic analysis of Hordeum marinum Huds. based on nucleotide sequences linked to the vrs1 locus

 More than 100 accessions of Hordeum marinum ssp. marinum, ssp. gussoneanum 2x and ssp. gussoneanum 4x were studied by analyzing the nuclear DNA homologous to the chloroplast translation elongation factor. PCR amplification using the locus-specific primers generated a 932 bp fragment from ssp. marinum, a 915 bp fragment from ssp. gussoneanum 2x, and 915 bp and 931 bp fragments from ssp. gussoneanum 4x. Sequencing of the entire DNAs showed that the 915-bp fragments of diploids and tetraploids are identical, indicating that the diploid probably is the immediate donor of the 915-bp fragment of tetraploids. Although the donor of the 931-bp fragment was not determined, phylogenetic analysis of the data showed that the clade of the 931-bp fragment was included in the H. marinum complex. A MspI site was diagnostic to distinguish ssp. marinum into two clades with disjunct distributions, the Iberian Peninsula (type 2) and other regions (type 1). Nucleotide variation was revealed in the type 2 accessions but all the other clades were highly uniform. Received January 3, 1999 Accepted January 24, 2001     

Fine mapping and candidate gene analysis of dense and erect panicle 3, DEP3, which confers high grain yield in rice (Oryza sativa L.)

Architecture of the rice inflorescence, which is determined mainly by the morphology, number and length of primary and secondary inflorescence branches, is an important agronomical trait. In the current study, we characterized a novel dense and erect panicle (EP) mutant, dep3, derived from the Oryza sativa ssp. japonica cultivar Hwacheong treated with N-methyl-N-nitrosourea. The panicle of the dep3 mutant remained erect from flowering to full maturation, whereas the panicle of the wild type plant began to droop after flowering. The dep3 mutation also regulated other panicle characteristics, including panicle length, grain shape and grain number per panicle. Anatomical observations revealed that the dep3 mutant had more small vascular bundles and a thicker culm than wild type plants, explaining the EP phenotype. Genetic analysis indicated that the phenotype with the dense and EP was controlled by a single recessive gene, termed dep3. The DEP3 gene was identified as the candidate via a map-based cloning approach and was predicted to encode a patatin-like phospholipase A2 (PLA2) superfamily domain-containing protein. The mutant allele gene carried a 408?bp genomic deletion within LOC_Os06g46350, which included the last 47?bp coding region of the third exon and the first 361?bp of the 3??-untranslated region. Taken together, our results indicated that the patatin-like PLA2 might play a significant role in the formation of vascular bundles, and that the dep3 mutant may provide another EP resource for rice breeding programs.     

An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA

In this study, we applied insertional mutagenesis using Agrobacterium transfer DNA to functionally characterize the gene of Brassica rapa L. ssp. pekinensis. The specific objectives were to: (i) develop and apply a gene tagging system using plasmid rescue and inverse PCR, (ii) select and analyze mutant lines, and (iii) analyze the phenotypic characteristics of mutants. A total of 3,400 insertional mutant lines were obtained from the Chinese cabbage cultivar, ’seoul’, using optimized condition. Plasmid rescue was performed successfully for transgenic plants with multiple T-DNA insertions, and inverse PCR was performed for plants with a single copy. The isolated flanking DNA sequences were blasted against the NCBI database and mapped to a linkage map. We determined the genetic loci in B. rapa with two methods: RFLP using the rescue clones themselves and sequence homology analysis to the B. rapa sequence database by queries of rescued clones sequences. Compared to wild type, the T₁ progenies of mutant lines showed variable phenotypes, including hairless and wrinkled leaves, rosette-type leaves, and chlorosis symptoms. T-DNA inserted mutant lines were the first population that we developed and will be very useful for functional genomics studies of Chinese cabbage.     

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incanaR. Br. (Brassicaceae)

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.     

Characterization of low-molecular-weight glutenin subunit genes from Hordeum brevisubulatum ssp. turkestanicum

Three novel low-molecular-weight glutenin subunit (LMW-GS) genes (designated as Ht1, Ht2, and Ht3) were isolated from the genomic DNA of Hordeum brevisubulatum ssp. turkestanicum by PCR amplification (accession no. Y0695). The coding regions of Ht1, Ht2, and Ht3 were 924, 924, and 903 bp, respectively. The deduced amino acid sequences were 306, 306, and 299 amino acid residues each with a signal peptide, a central repetitive region rich in proline and glutamine, and N-and C-terminal non-repetitive domains. A comparison was carried out of these genes with other known B hordein genes from cultivated barley and LMW glutenin genes from wheat. The results indicated that Ht1, Ht2, and Ht3 had a more similar structure and a higher level of homology with the LMW-GS genes than the B hordein genes. In order to investigate the evolutionary relationship of the novel genes with the prolamin genes from barley and wheat, the phylogenetic tree was constructed and the subfamilies of these prolamin genes were identified. The results suggested that the three novel genes were glutenin-like proteins designated as LMW-m type genes. The text was submitted by the authors in English.     

Characterization of a pollen-preferential gene, BAN102, from Chinese cabbage

We isolated and characterized a pollen-preferential gene, BAN102, from Chinese cabbage and analyzed the activity of its promoter. There were three or four copies of the BAN102 gene in the Chinese cabbage genome that specifically expressed in pollen and pollen tube. There were 2137 bp of BAN102 genomic clone comprising 186 bp of protein coding region, and 1178 bp of 5′ and 773 bp of 3′ non-coding regions. TATA box were located at 1071 nt of the promoter region while the polyadenylation signal and polyadenylation site were at 1470 and 1486 nt of the 3′ non-coding region. BLAST search of BAN102 sequence showed that coding region of BAN102 gene was the greatest percent similarity with arabinogalactan protein (AGP23) gene from Arabidopsis thaliana. Promoter analysis using GUS gene as a reporter showed that the pollen-specificity of BAN102 resided within the −112 to −44 bp of proximal promoter from the transient expression in tobacco and Chinese cabbage plants.     

雄性不育相关基因msLTP的反义RNA验证

根据普通白菜雄性不育相关的脂质转移蛋白基因(msLTP)的cDNA序列设计引物,从普通白菜花蕾的cDNA中扩增出312bp的片段,然后将该片段连接至双元载体pBI12l中,得到反义RNA植物表达载体并导入农杆菌LBA4404菌株中,通过农杆菌介导法转化菜心;利用PCR和Southern blot分析检测得到了25株转基因植株,转基因植株的花粉部分畸形或空瘪,花粉离体萌发率为38.56%,较未转化植株的萌发率(76.32%)降低了37.76个百分点.研究表明,反义RNA技术使msLTP基因沉默而导致了菜心转基因植株的部分花粉发育不良,说明msLTP基因在普通白菜和菜心等花粉发育中具有重要作用.     

Molecular evidence for post-domestication selection in the <Emphasis Type="Italic">Waxy</Emphasis> gene of Chinese waxy maize

Waxy maize was first reported in China in 1909 and is mainly used in food production in Asia. The evidence for strong domestication selection in the Waxy locus of rice and a selective sweep around its genomic region make us to wonder whether there has been similar selection in Waxy in glutinous maize. To address this issue, DNA sequences of Waxy, three flanking genes and an unlinked gene (Adh1) of 30 accessions sampled from Chinese waxy maize accessions, including representative landraces and inbred lines, were determined in this study. Sharp reduction of nucleotide diversity and significant neutrality tests (Tajima’s D and Fu and Li’s F*) were observed in the Waxy locus in Chinese waxy maize but not in nonglutinous maize; comparison with the unlinked gene confirmed that this pattern was different to Waxy. Sequence analysis across a 143 kb genomic segment centered on the Waxy locus revealed patterns consistent with a selective sweep in the upstream region of Waxy. The selective sweep detected based on current limited genomic sequences exceeded over 50 kb, indicating strong selection in this or a bigger region. However, No sweep effect was detected in the repetitive downstream region of Waxy. Phylogenetic analysis indicated that Chinese waxy maize was domesticated from the cultivated flint maize (Zea mays ssp. mays) that was introduced from the new world. At least two independent deletions in exon 7 (30 bp) and 10 (15 bp) were identified in the Chinese accessions respectively. These findings demonstrate a similar pattern of domestication selection in the Waxy genomic region in both glutinous maize and rice, suggesting that this pattern in the rise of glutinous phenotype is likely in other cereal crops.     

Cloning of the cDNA and genomic clones for glutathione synthetase fromArabidopsis thaliana and complementation of agsh2 mutant in fission yeast

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated fromArabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast,Schizosaccharomyces pombe, and shared only a small region of similarity with theEscherichia coli protein. A 4.3 kbSstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons.When theArabidopsis cDNA cloned in a special shuttle vector was expressed in aS. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in thegsh2 ^- mutant, and restored to substantial levels by the expression of theArabidopsis cDNA. TheS. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols,¹⁰⁹Cd²⁺ binding activity, and cadmium resistance. Since theArabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.     

沙枣叶绿体全基因组序列及其使用密码子偏性分析

该研究以2株野生沙枣(Elaeagnus angustifolia Linn.)嫩枝经温室水培后的嫩叶为材料,采用CTAB法分别提取总DNA,并利用第二代测序技术进行总DNA从头测序,组装后得到2株沙枣叶绿体基因组全序列,并详细分析了其蛋白质编码基因密码子使用的偏好性及其原因,为沙枣叶绿体基因工程和分子系统进化等研究奠定基础。结果显示:(1)组装得到沙枣叶绿体基因组序列全长150 546 bp,由长度为81 113 bp的长单拷贝(LSC)区域和25 494 bp的短单拷贝(SSC)区域,以及1对分隔开它们的长18 445 bp的反向重复序列(IRS)组成;注释共得到132个基因,包括86个蛋白编码基因、38个tRNA基因和8个rRNA基因。(2)沙枣叶绿体基因组蛋白编码基因密码子的第三位碱基GC含量(GC_3)为28.47%,明显低于整个叶绿体基因组GC含量(37%),也低于第一位(GC_1)和第二位(GC_2)碱基的GC含量,说明密码子对AT碱基结尾有偏好性;其中, UCU、CCU、UGU、GCU、CUU、GAU、UCA和UAA为最优密码子。(3)同义密码子相对使用频率(RSCU)分析发现,影响密码子使用模式的因素并不单一,密码子的偏好性受到突变、选择及其他因素的共同影响,并且自然选择表达引起的序列差异比突变对密码子偏好性的影响要显著;中性绘图分析、有效密码子数(ENC-plot)分析和奇偶偏好性(PR2-plot)分析表明,沙枣叶绿体基因组使用密码子的偏性受选择的影响更大。(4)通过最大似然法、最大简约法和贝叶斯方法对胡颓子科6个物种和1个枣的叶绿体基因序列构建系统发育树,与它们使用密码子偏性聚类的结果一致,表明叶绿体基因组使用密码子偏性与物种的亲缘关系相关。     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

Cloning and characterization of G+C-rich, highly repeated DNA sequences from Porphyra yezoensis (laver), Rhodophyta

G+C-rich sequences in the genomic DNA of Porphyrayezoensis (laver) were cloned and characterized. Sequence analyses of the genomic DNA inserted in fiveclones showed that the DNA contained long G+C-richstretches of more than 200 bp. These stretchesconsisted of more than 80% G+C residues. TheG+C-rich sequences were highly repeated andinterspersed throughout the genome of P.yezoensis and constituted about 6.0–6.6% of thegenome. Parts of these sequences were tandem repeatedin arrays. Hybridization experiments showed thatthese highly repeated, interspersed G+C-rich sequenceswere present in other species of Porphyra, butnot in species of the genera Grateloupia and Gelidium, suggesting that these sequences mightevolve rapidly among genomes, species and genera.     

Disruption of a <Emphasis Type="Italic">fur</Emphasis>-like gene inhibits magnetosome formation in <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> MSR-1

In this study, a genomic library of Magnetospirillum gryphiswaldense MSR-1 strain was constructed and a fur-like gene (encoding Fur protein, ferric uptake regulator) was isolated and sequenced. This gene consisted of 420 bp and encoded 139 amino acid residues. To investigate the function of this gene in MSR-1, a fur mutant was generated by double crossover with a kanamycin cassette inserted into its coding region. Iron uptake and magnetosome formation were dramatically inhibited by disruption of fur. Iron content analysis of the fur mutant indicated that it contained approximately 0.037% by dry weight, which was at least 10-fold less than that observed in the wild type. Electron microscopy revealed the absence of a magnetosome in the fur mutant, although it was able to tolerate 1 mM H₂O₂ at 10-fold higher level than wild-type. These data suggest that Fur protein may possess a novel function in magnetic bacteria. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 11, pp. 1532–1539.     

Pea polyubiquitin genes: (I) structure and genomic organization

Four polyubiquitin genes, PUB1, PUB2, PUB3 and PUB4, were isolated from a pea (Pisum sativum L. cv Alaska) genomic library and completely sequenced. They represent all of the four polyubiquitin genes of the ubiquitin gene family in pea. The coding regions of the genes contain five or six coding units arranged as tandem repeats. The different coding repeats of the four genes share homologies between 75 and 97%, encoding the same protein of 76 amino acids identical to those from other higher plants. The open reading frames of PUB1, PUB2 and PUB4 terminate in the additional amino acid, phenylalanine (F), and PUB3 terminates in isoleucine (I). The polyubiquitin genes all contain intron sequences ranging from 584 to 1114 bp immediately 5′ to the ATG initiation codon of the first coding sequence. Of the four genes, three are associated with long AT-rich (85.4–89.4% A+T) sequences ranging from about 331 to 478 bp at their 5′ or 3′ ends. The PUB4 gene was found to be linked to a moderate to highly repetitive DNA at its 5′ flanking sequence. The greater sequence homology between different genes than among individual repeating units of a gene suggests that the polyubiquitin genes may have arisen by gene duplication of a single gene sequence.     

虎杖叶绿体基因组结构与变异分析

虎杖(Reynoutria japonica Houtt.)为蓼科(Polygonaceae)蓼族(Polygoneae)虎杖属(Reynoutria Houtt.)植物,是一种传统的中草药,具有利湿退黄、清热解毒、散瘀止痛、止咳化痰的功效。本研究采取高通量测序技术获得5个虎杖品种的叶绿体全基因组序列,并与NCBI已公布的蓼族何首乌(Fallopia multiflora)和金线草(Antenoron filiforme)等植物的叶绿体全基因组序列进行了基因组学和系统发育分析。通过基因组学分析发现,5种虎杖的叶绿体基因组大小有163 376 bp和163 371 bp两种情况,并呈现出典型的环状四分体结构,85 784 bp的一条较长的单拷贝区(large single-copy region,LSC),18 616 bp的一条较短的单拷贝区(small single-copy region,SSC),还有两条长度一致的反向重复区,分别为IRa区和IRb区相间隔分布。通过注释得到161个基因,其中蛋白编码基因106个,rRNA编码基因10个,tRNA编码基因45个。总GC含量为36.7...