RESUMO
For the sake of screening novel genes related to the male sterility in chili pepper for studying the molecular mechanism of plant male sterility, the gene differential expression analysis was performed by cDNA-amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile-fertile line 114AB of Capsicum annum L., and a variety of differentially expressed cDNA fragments were detected in fertile or sterility material. Camf1, a transcript-derived fragment (TDF) accumulated in fertile line flower buds was further investigated. The Camf1 has 1,854 bp in length with no introns containing a 1,707-bp open reading frame (ORF). The deduced amino acid sequence of Camf1 shares higher similarity to some members from a glyoxal oxidase-related protein family, and has a glyoxal oxidase conserved domain at the N-terminus and a domain of unknown function (DUF1929) at C-terminal end. Expression analysis showed that Camf1 expressed only in stage 3-7 flower buds of male fertile of C. annum L. 114AB, and no detection in all organs of male sterility. The peak of expression level of Camf1 appeared at stage 4 flower buds of fertile line. Furthermore, expression analysis of different organs revealed that Camf1 expressed only in anthers of male fertile material and there were no expression in sepals, petals, pistils, roots, stems, leaves and open flowers. These results suggested that Camf1 was an anther-specific gene and might be essential for the fertility of C. annum L.
Assuntos
Capsicum/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/genética , Infertilidade das Plantas/genética , Oxirredutases do Álcool/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Sequência de Bases , Capsicum/fisiologia , Biologia Computacional , Primers do DNA/genética , DNA Complementar/genética , Fertilidade/genética , Componentes do Gene , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Infertilidade das Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
In order to find the best screening kanamycin concentration in the genetic transformation of mustard (Brassica juncea Coss.), the seedling cotyledons of mustard were placed on bud-induced media supplemented with different kanamycin concentrations. The bud differentiation of explants was totally inhibited when the kanamycin concentration was greater than 30 mg/L. The seeds of mustard were placed on germination media supplemented with different concentrations of kanamycin. All the seedlings were white when kanamycin concentration was higher than 200 mg/L. The leaves of mustard in field were smeared with the solutions including different concentrations of kanamycin. The treated leaves showed white when kanamycin concentration was over 200 mg/L. To study the segregation of the alien gene in transgenic mustard offspring, the transgenic mustard seeds and the leaves of the transgenic mustard offspring using npt-gene as assistant selection-marker were treated with 200 mg/L kanamycin, and the results were analyzed by chi-square test. The segregation ratio in the offspring of 4 transgenic lines with single copy of transgene agreed with a ratio of 3:1. The segregation ratio in offspring of the one transgenic line with double copies was agreed with a ratio of 3:1, and the segregation ratio in offspring of the other transgenic line with double copies was agreed with ratios of 3:1 and 15:1 simultaneously. It is indispensable to thoroughly study the insert of the double copies of transgenes in transgenic mustard. PCR technology was used to confirm the above detection methods. It is concluded that applying kanamycin to screen transgenic mustard offspring is feasible.
Assuntos
Canamicina/farmacologia , Mostardeira/efeitos dos fármacos , Mostardeira/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Cotilédone/efeitos dos fármacos , Cotilédone/genética , Germinação/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase , Sementes/efeitos dos fármacos , Sementes/genéticaRESUMO
A cDNA library was constructed from cabbage 84075 which resists TuMV. The degenerate primers was designed with the disease resistance gene conservative domain (NBS-LRR). The fragments of 513 bp were amplified by RT-PCR and genomic DNA PCR from resistant material 84075, then cloned and sequenced. Two recombinants which are highly homologous with the resistance genes cloned in other plants were used as probes, (named as Bor1, Bor2), the cDNA library was screened. A positive clone was obtained, named as TuR2, whose length was 762 bp, which encodes 226 amino acids, contains a long 681 bp open reading frame (ORF), and has different homology score of amino acid compared with the cloned resistance disease genes of other plants. TuR2 was used as probe. Southern blotting hybridization with genomic DNA shows that TuR2 is probably present in single copy; No. rthern blotting hybridization with RNA shows that the gene expression is constitutive and not differential in every part of the resistance plant 84075, the result of separating detection in F2 population shows that TuR2 gene is probably related to resistance to TuMV in cabbage.