水稻BSL基因研究进展

综述了水稻基因图位克隆的一般策略 ,水稻BSL基因研究的进展 ,以及水稻BSL基因的应用前景。

Fine Mapping and Isolation of Bc7(t),Allelic to OsCesA4

Several brittle culm mutants of rice were identified and characterized. In this study, we characterized a brittle mutant （bc7（t）） identified from japonica variety Zhonghua 11 by means of ^60Co-γ radiation. This mutant displays normal phenotype similar to its wild type plants except for the fragility of all plant body, with - 10% decrease in the cellulose content. The genetic analysis and gene fine mapping showed that the bc7（t） mutant was controlled by a recessive gene, residing on an 8.4 kb region of the long arm of chromosome 1. The gene annotation indicated that there was only one putative gene encoding cellulose synthase catalytic subunit （CesA） in this region, which was allelic to OsCesA4. Furthermore, the sequence analysis was carried out and 7 bases deletion in the junction of exon 10 and intron 10 was done in bc7（t） mutant, resulting in the change of reading frame and the consequent failure to generate functional protein. In addition, the result of RNA interference experiment showed that when the Bc7（t） was knocked down, the transplants exhibited fragility, similar to bc7（t） mutant. The finding of novel allele of OsCesA4 locus will facilitate the understanding of the mechanism of cell wall biosynthesis. The potential utilization of the bc7（t） mutant in animal food was discussed as well.

Cloning of the APRT Gene from Rice and Analysis of Its Association with TGMS

Adenine phosphoribosyltransferase (APRT) is the major enzyme that converts adenine into adenosine-3'-phosphate (AMP). APRT-deficient mutant caused by APRT gene mutation results in the male sterility in Arabidopsis thaliana L. In order to confirm the existence of rice APRT gene and to investigate its association with thermo-sensitive genic male sterile (TGMS) phenotype of rice, a APRT gene was identified from BLAST search of the rice genome database using APRT gene sequences from other plant species as probes. Further, the gene was cloned from rice and named APRT(GenBank accession number AY238894) using the combination of bioinformatic and experimental approaches. The rice APRT was located in the 56 000 bp to 63 000 bp region of a rice bacterial artificial chromosome (BAC) clone (AL606604) on chromosome 4 and was deduced by software from the positive DNA clone. Its cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed according to the sequence of the putative gene. The full-length cDNA was obtained by rapid amplification of cDNA ends (RACE) procedure and was sequenced. Open reading frame (ORF) analysis indicated that the rice APRT gene encodes a peptide of 212 amino acid residues, including seven exons and six introns. Using reverse position specific BLAST (RPS-BLAST), the APRT domain was identified in the polypeptide. The homology comparison demonstrated that the polypeptide exhibits 54.9%, 54.9%, 49.6% and 59.5% identity with that from Hordeum vulgare, Ttriticum aestivum, and A. thaliana (APRT types 1 and 2), respectively. Comparing the sequence of APRT gene from TGMS mutant lines 'Annong S-1' (Oryza sativa subsp. indica) with that from its corresponding wild type 'Annong F' (Oryza sativa. subsp. indica), we found that there are five single nucleotid polymorphism (SNP) sites in the gene of 'Annong S-1', which locate mainly in the second intron. However, the result of cDNA sequencing showed that these SNP sites do not damage the successful splicing of intron 2. Qualitative RT-PCR and Northern blot indicated that the gene tran-scription in the 'Annong S-1' young panicles that were verified to be the thermo-sensitive organ at the early stage of pollen fertility alternation is down-regulated by high temperature stress (28 V), which is the critical temperature causing 'Annong S-1' fertility conversion. These results revealed that the change of expression pattern of APRT in young particles of 'Annong S-1' in high temperature conditions is perhaps related to the TGMS of 'Annong S-1'.

Cloning and Analysis of ISA1 from Oryza sativa

[Objective] The aim was to clone ISA1 from Oryza sativa and analyze its expression situation in different tissues and different endosperm filling stage.[Method] With japonica rice cultivar nipponbare as test material,the expression patterns of ISA1 in different tissues and different endosperm filling stage was analyzed by using semi-quantitative RT-PCR technique.[Result]The full length open reading fragments of ISA1 encoded 811 amino acid residues.The homologous alignment and phylogenetic analysis showed th...

Cloning and Expression Pattern Analysis of Nitrogen- Starvation-induced Genes in Rice

To understand the regulation system of nitrogen X-starvation in higher plants, a cDNA library from N-starved rice (Oryza sativa L.) seedlings was constructed using rapid subtraction hybridization (RaSH) procedure. Through reverse Northern analysis and Northern blotting, 18 unique known genes and two unique unknown genes were identified, which were up-regulated by N-starvation in rice. The known genes are involved in several metabolisms including carbon metabolism, secondary metabolite synthesis, ubiquitylation and protein degradation, phytohormone metabolism, signal transduction, growth regulator and transcription factors. Different induced expression patterns based on spatial and temporal express ions were found for these genes. The results indicate the cross-talks between N-starvation response and various metabolisms in plants.

Cloning and Analysis of WRKY Gene of Rice Induced by Rhizoctonia solani Kuhn

[Objective] The aim was to clone the up-regulated expression gene of rice induced by Rhizoctonia solani.[Method] The EST fragment K16 obtained by suppression subtraction hybridization（SSH）was cloned and confirmed by reverse transcription-polymerase chain reaction（RT-PCR）.Then RT-PCR products were cloned into the PMD18-T vector and sequenced.The functions of the sequence were predicted with bioinformatics method.[Result] A 1 079 bp gene was obtained.The gene encoded a protein with 236 amino acids.The protein contains many motif sites,two WRKY domains and a C2H2 zinc finger motif.The gene showed high identities with WRKY8,WRKY24 and WRKY30 gene of rice.[Conclusion] The up-regulated expression gene induced by R.solani was representative WRKY family gene.The gene could play an important role on rice sheath blight resistance.

Cloning and Genetic Transformation of OsOle1 Gene in Rice

[Objective] The cloning and transformation of rice OsOle1 gene were conducted in the research.[Method] OsOle1 gene was cloned by RT-PCR.The amplified OsOle1 was then ligased to pCAMBIA 1300 to construct GUS overexpression vector.Then the Agrobacterium-mediated method was used in rice callus transformation.[Result] The full-length of OsOle1 gene was 498 bp and it encoded 189 amino acids.The over-expression vector Ub：：OsOe1-GUS was prepared and transgenic plants were successfully obtained.[Conclusion] The transgenic lines laid the foundation for the function research of OsOle1.

A Cosmid Library Constructed to the Elite Rice Cultivar “Minghui 63”

A genomic DNA library was constructed to the elite rice cultivar “Minghui 63” using the cosmid SuperCos1 as the vector. The library consisted of 45000 clones with average insert size about 40 kb. It was estimated that this library had a capacity of 4.2 times equivalent of the haploid genome of rice.

Cloning and Expression Analysis of a Human Putative Tumor Suppressor Gene Homologue from Rice

A gene homologous to the human Putative tumor suppressor gone QM, designated OSQM1, was isolated from rice (Oryza sativa L.) genomic DNA library using through homology screening. It contained 4 exons and 3 introns, encoding a protein of 219 amino acids with 46 basic amino acids, leading to a high isoelectric point of 11.02. Homology search showed that this gene existed in eukaryotes and highly conserved throughout eukaryotes, suggesting an essential role of this gene. Northern Not analysis showed that it was expressed in various rice organs, but at lower level in developing flower and callus tissue than in other vegetative organs. Its expression levels in roots and leaves were influenced by different environmental factors.

Characterization and cloning of SMALL GRAIN 4, a novel DWARF11 allele that affects brassinosteroid biosynthesis in rice

Brassinosteroids （BRs） are important hormones that regulate plant development and physiology. Substantial progresses have been made in BR-related studies, and especially an increasing number of new genes involved in BR biosynthesis have been identified. Here, we characterize a BR-related rice mutant, small grain 4 （sg4）, obtained from callus culture of japonica cultivar Nipponbare. This mutant showed multiple phenotypes such as dark green, rugose erect leaves and small round grains. It was higher than the wild type, different from the majority of BR- and gibberellin-related mutants. Genetic analysis showed that the mutant phenotypes are controlled by a single recessive locus. The gene was fine-mapped to a 90.7-kb interval with 1,100 F2 recessive individuals by means of map-basedcloning. Totally 11 open reading frames were found in this interval, only one of which was detected with an 8-bp in- sertion in the 5rUTR region by sequencing. Functional complementation test revealed that a DWARFll allele, LOC_OsO4g39430, is answer for the mutant phenotype of sg4. Tissue-specific response to BR and decreased expression levels of BR biosynthetic genes suggest that sg4 is a weak BR-deficient mutant. These results are beneficial to understanding the physiological action of sg4 in a more comprehensive way.