An Intron Sequence is Present in the 3 UTR of Tomato Cf9 Gene

Tomato leaf mould-resistance gene Cf) was amplified by PCR and cloned from the genomic DNA of a tomato ( Lycopersicon esculentum Mill. ) cultivar ("Zhongza 9'). Sequence analysis indicates that this Cf9 gene is 2 751 bp long and contains an open reading frame encoding a protein of 863 amino acids. An intron of 115 bp in length is found in the 3' untranslated region of this gene. The exon/intron borders are replicate sequences, TCCAGG(T)ATTC, which shares high homology with those of the two genes of Cf2 which is another leaf mould-resistant gene locus in tomato. Compared with the previously reported cDNA sequence of Cf9, a single change of T to C occurs at the nucleotide position of 371 in this PCR-ampliiied Cf) gene, which converts Leu 121 to Pro in the LRR region of the encoded protein.     

Structure and expression of a rice hsp70 gene

A genomic hsp70 gene was isolated from a rice IR36 genomic library and 4 794 bp of the gene have been sequenoed. The 5' flanking region of the gene contained a putative TATA box and a typical heat shock element sequence 5'-CTcgGAAccTTCgAG-3'. The amino acid sequence of the rice HSP70 deduced from the coding region shared 84%-92% homologies with those of HSP70s from other plant species. An intron 1939bp long was identified in the coding region at the codon specifying amino acid 72 (Asp), the similar position introns occurring in other intron-containing hsp70 genes. In addition, another intron of 57 bp was found in the 3'-untranslated region in the rice hsp70 gene. Southern blot hybridization showed that rice hsp70 gene family contained at least three members. Analysis of the RNA leveis with the gene-specific and non-specific probes revealed that the rice hsp70 gene expressed at normal temperature and the expression was enhanced by heat shock treatment.     

梅花‘南京红须’F3'H全长基于gDNA的TAIL-PCR法克隆

根据从基因组DNA扩增到的梅花‘南京红须’类黄酮3’-羟化酶基因片段（469bp）设计3条嵌套的特异性引物．与6条短的随机简并引物组成的引物库分别用热不对称交错PCR法从‘南京红须’基因组DNA扩增该片段的5’和3’旁侧序列。获得的5’和3’旁侧序列分别长1443bp和1200bp。将两个旁侧序列在469bp片段的基础上拼接得到‘南京红须’全长为2lrl4bp的类黄酮3’-羟化酶基因,被命名为pmhxF3’H。序列分析表明：该基因与11条正式发表的、已递交到GenBank的类黄酮3’-羟化酶基因的eDNA序列在总体上有52．21％的一致性．具有3个内含子。其启动子含有1个“AGGA盒”、1个“GC盒”和3个“TATA盒”。这是首次用热不对称交错PCR法从木本植物的基因组DNA克隆到类黄酮3’-羟化酶基因。本研究将为梅花花色的分子生物学机理探索、花色的基因工程改良提供参考。     

Development of a STS marker assay for detecting loss of heterozygosity in rice hybrids.

The RAPD (random amplified polymorphic DNA) markers OPE15750 and OPE15300 were affected by loss of heterozygosity (LOH) in rice hybrids AMR x 'M202' and AMR x 'L202'. The markers were mapped to the same locus at or near the centromere of rice chromosome 2. The two RAPD products were cloned, sequenced, and found to have lengths of 734 bp and 297 bp, respectively. The 297-bp sequence shares a 98% homology with one end of the 734-bp sequence, accounting for the cross-hybridization previously observed between the two clones. Based on the DNA sequence of the 734-bp fragment, a pair of STS (sequence-tagged site) primers was designed and tested. Rice plants homozygous for either OPE15734 or OPE15297 all produced PCR fragments of the same length, 482 bp. However, the two PCR products were discernible by digestion with the restriction enzyme XbaI prior to gel electrophoresis. The STS product from plants homozygous for OPE15734 was cut into two fragments of 239 and 240 bp, which appeared as one single band in an agarose gel; whereas the STS product from plants homozygous for OPE15297 was not cut by XbaI and was characterized by a 482-bp band in the agarose gel. These STS primers were tested in rice hybrids and F2 progenies derived from the hybrids of AMR x 'M202' and AMR x 'L202'. Homozygosity for OPE15297 was confirmed for all F2 panicle rows derived from AMR x 'M202'. In contrast, F2 panicle rows of AMR x 'L202' exhibited two different segregation patterns (genotypes), i.e., either uniformly homozygous for the 240-bp marker (OPE15734/OPE15734) or segregating for the 482- and 240-bp markers (OPE15734/OPE15297). This STS-marker system provides a robust assay for detecting the occurrence of LOH in rice hybrid progenies.     

野生稻和栽培稻的随机多态DNA(RAPD)分析

应用 RAPD方法对药用野生稻、普通野生稻、粳稻和籼稻进行基因组多态性分析。 1 2个随机引物共扩增出 1 3 2条 RAPD带 ,片段大小在 3 0 0～ 3 5 0 0 bp之间 ,其中有 1 0 6条表现出多态性 ,占总扩增片段的86.4%。根据遗传距离分析 ,用 UPGMA法构建了聚类树状图 ,结果表明 ,普通野生稻的遗传特性比药用野生稻更接近于栽培稻。     

Selection of RAPD marker for growth of seedlings at low temperature in rice.

We have developed a polymerase chain reaction (PCR)-based assay that could effectively reduce the time period required to screen and select the cold tolerance gene of rice seedlings under field conditions. The two specific random amplified polymorphic DNA (RAPD) fragments for the assay were identified on the basis of quantitative trait loci (QTL) analysis which were found to be tightly linked to cold sensitivity. The two RAPD fragments, OPT8(600) in the cold sensitivity rice cultivar 'Dular (indica)' and OPU20(1200) in the resistance rice cultivar 'Toyohatamochi (japonica)', were identified after screening 11 RAPD fragments using 2 random primers on the genomic DNAs of 'Dular' and 'Toyohatamochi'. These primers, when used in a multiplexed PCR, specifically amplified a 0.6 kb and a 1.2 kb fragment in the sensitive and resistant rice cultivars, respectively. When this assay was performed on the genomic DNAs of 16 japonica, 3 Tongil (indica/ japonica), and 2 indica rice cultivars, the primers amplified a 0.6 kb fragment in all of the cold sensitivity rice cultivars or 1.2 kb fragment in all of the resistance ones. These markers can be of potential use in the marker-assisted selection (MAS) for cold tolerance in rice seedling. As screening for resistance can now be conducted independent of the availability of low temperature, the breeding of cold tolerance cultivars can be hastened.     

Analysis of OPT8511 RAPD fragments closely linked with cold sensitivity at seedling stage in rice (Oryza sativa L.)

OPT8(511) was confirmed to be strongly associated with cold sensitivity of rice by random amplified polymorphic DNA (RAPD) analysis for the cold tolerance with 94 F2 population crossed with 'Dular' (cold sensitive cultivar) and 'Toyohatamochi' (cold resistant cultivar). A DNA marker from the RAPD fragment, OPT8(511), has been cloned with genomic DNA from rice cultivar ('Dular') and the nucleotide sequence has been determined. The nucleotide sequence revealed that the putative open reading frame was 511 base pairs and contained 169 amino acid residues. It is 79% and 57% identical to the rice cDNA (C26347) in DataBank at the nucleotide and amino acid sequence levels, respectively. The clone OPT8(511) specifically amplified a 511 bp band from the DNA of cold sensitive cultivars. Use of this marker could facilitate early selection of character associated with cold tolerance in rice.     

细胞质雄性不育辣椒育性恢复基因特异分子标记的筛选

利用集团分离分析法(Bulked segregant analysis BSA),以辣椒细胞质雄性不育系BU-12、恢复系RF-12为材料共筛选了336条RAPD引物,其中引物S418在恢复系中呈现特异性扩增,得到一条约3000bp的特异片段。回扩得到两条片段,测序表明大小为1515bp,1162bp。荧光原位杂交证实1515bp片段为恢复系特有,命名为S418_(1515)。序列分析表明S418_(1515)为一新发现的序列,Blastn序列比对同源性小于40%,tBlastx比对发现该序列与水稻2、4、7、10号染色体的几个BAC克隆上的序列高度同源。推测可能与其具有相似的编码功能,为进一步从分子水平研究辣椒育性恢复打下了坚实的基础。根据测序结果设计特异引物,将S418_(1515)转化成特异PCR标记,证明能用于候选材料的初筛。     

细胞质雄性不育辣椒育性恢复基因特异分子标记的筛选

利用集团分离分析法(Bulked segregant analysis BSA),以辣椒细胞质雄性不育系BU-12、恢复系RF-12为材料共筛选了336条RAPD引物,其中引物S418在恢复系中呈现特异性扩增,得到一条约3000bp的特异片段。回扩得到两条片段,测序表明大小为1515bp,1162bp。荧光原位杂交证实1515bp片段为恢复系特有,命名为S4181515。序列分析表明S4181515为一新发现的序列,Blastn序列比对同源性小于40％,tBlastx比对发现该序列与水稻2、4、7、10号染色体的几个BAC克隆上的序列高度同源。推测可能与其具有相似的编码功能,为进一步从分子水平研究辣椒育性恢复打下了坚实的基础。根据测序结果设计特异引物,将S4181515转化成特异PCR标记,证明能用于候选材料的初筛。     

Molecular Mapping of Rice Blast Resistance Gene Pi-k h in the Rice Variety Tetep

We have used rice line Tetep as a resistant donor with the aim of mapping a durable blast resistance gene Pi-k ^h using RAPD and AFLP techniques in conjunction with bulk segregant analysis. An F₂ mapping population consisting of 205 plants was generated by crossing Tetep with HP2216, a highly susceptible cultivar. Inoculation with specific isolate (PLP-1) of Magnaporthe grisea at seeding stage showed that the Pi-k ^h gene inherited as a single dominant gene in F2 population. RAPD analysis was performed with 240 primers to detect polymorphism between resistant and susceptible parents. Of these, 48 primers produced polymorphic banding pattern between resistant and susceptible parents. Bulk segregant analysis was performed with 48 primers of which 5 showed polymorphism between resistant and susceptible bulks. A 700 bp DNA band was obtained in resistant F2 plants with primer 5-129 indicating its linkage to the resistance gene. Out of 64 AFLP primer combinations used for polymorphism survey between HP 2216 and Tetep, 11 AFLP primer combinations were able to distinguish the resistant and susceptible bulks. An AFLP band of 75 bp obtained with primer combination, E-TAlM-CTC co-segregated with the resistance gene. The RAPD marker 5-129₇₀₀ and AFLP₇₅ were placed on the linkage map at a distance of 2.1 eM and 15.1 eM flanking to Pi-k ^hgene, respectively. The RAPD band closely linked to Pi-k ^h gene was sequenced and used for the development of CAPs markers which also co-segregated with resistant phenotype in the mapping population. On sequence analysis and homology search of RAPD fragment with whole rice genome sequence database and the information available on physical, genetic and sequence maps of rice, the co-segregating CAPs marker was placed at long arm of rice chromosome 11. CAPs marker developed in this study showed polymorphism in different rice cultivars grown in North-Western Himalayan region and is being used for the pyramiding of Pi-k ^h gene along with other blast resistance genes using marker-assisted selection.     

Rapid PCR-based determination of transgene copy number in rice

We present a simple, rapid, and low-cost method to determine transgene copy number in rice. More than 100 first- and second-generation transgenic rice plants were tested. The plasmid (pRCopy) used for rice transformation contains the specific gene of interest and a partially deleted cytochrome c gene (cyc), a single-copy gene in rice. A 132-bp segment of the cloned ricecyc was shortened to 108 bp by deleting a 24-bp internal fragment. After PCR amplification of the genomic DNA from transgenic rice harboring pRCopy, the 2 expected bands were found. The 121-bp band corresponds to the endogenouscyc; the 97-bp band comes from the integrated pRCopy. Clear distinctions can be made between single and multiple copies of the transgene by comparing band densities.     

Heteroduplex molecules formed between allelic sequences cause nonparental RAPD bands.

Primers (10-mers) of random sequence were used to amplify RAPD bands from genomic DNA of an F1 strain of flax rust (Melampsora lini) and its two parent strains. One primer out of 160 tested was unusual in that it amplified a product from F1 DNA that was not amplified from either parental DNAs. The same primer also generated two RAPD bands that segregated as codominant alleles amongst F2 progeny. The nonparental band was only generated from DNAs of F2 individuals that were heterozygous for these two allelic sequences. Sequence analysis of the two RAPD alleles demonstrated greater than 99% sequence identity, although the larger allele possessed an additional 38bp relative to the smaller. Mixing of the two allelic sequences followed by denaturation and annealing in the absence of polymerase activity resulted in the formation of the nonparental band. Thus the nonparental band present in some RAPD reactions consisted of a heteroduplex molecule formed between two allelic sequences of different size. These data demonstrate that heteroduplex molecules formed between allelic RAPD products are a potential source of artifactual polymorphism that can arise during RAPD analysis.     

A partial sequence of lipoxygenase gene from genomic DNA of aromatic rice (Oryza sativa L.)

Aromatic (Bas-370, PB-1) and non-aromatic (Pusa-677, Pusa-834) rice were selected for the characterization and for distribution of lipoxygenase (Lox) genes. Polymorphism was observed when genomic DNA of rice varieties was hybridized with a heterologous lipoxygenase probe. A distinct polymorphic fragment (approximately 1.2 kb) was found in Bas-370. Sub-genomic library of Bas-370 was constructed and screened with LoxA probe. The smallest putative clone (pBas-14) of approximately 1.2 kb was sequenced. Complete nucleotide and deduced amino acid sequence showed the clone was 1134 bp long and comprised of 378 amino acid residues. PCR amplification of genomic DNA from four rice varieties with a soybean Lox primer also showed a polymorphic fragment of size approximately 600 bp (amplicon) in aromatic varieties that was sequenced directly. Nucleotide sequence alignment between pBas-14 and amplicon concluded that the amplicon was a part of the insert pBas-14.     

Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L.

Interleukin (IL)-10 was cloned from the common carp (Cyprinus carpio L.) using IL-10 primers from carp head kidney following stimulation with concanavalin A and lipopolysaccharide. The cDNA consisted of a 1096 bp sequence containing a 55 bp 5' untranslated region and a 498 bp 3' untranslated region. An open reading frame of 543 bp encoded a putative 180 amino acid protein with a putative signal peptide of 22 amino acids. The signature motif of IL-10 is conserved in carp sequence. A 2083 bp genomic sequence of carp IL-10 was found to contain five exons interrupted by four introns. With the exception of much more compact introns, the genomic structure was similar to that of mammalian IL-10. By homology, phylogeny and genomic analyses, the carp gene cloned was designated as IL-10. Carp IL-10 was expressed in head, kidney, liver, spleen and intestine during the resting phase. The gene was also expressed in head kidney and liver following in vitro stimulation with lipopolysaccharide.