Studies On Differentially Expressed Genes Of Genic Male Sterile Two-type Line In Zinnia Elegans

Youth-and-old-age is an annual plant that belongs to genus Zinnia of Asteraceae family. It is widely cultivated in China, especially for flower beds, clusters and borders. Recessive genic male sterility (RGMS) is used to produce F1seeds because of various remarkable advantages, such as its stable and complete male sterility and no negative cytoplasmic effect on yield. However, we still have little knowledge about the mechanism of its fertility control. So it is necessary to do some researches about the mechanism of fertility control for better utilization of the RGMS.In the present study, the fertile plants and sterile plants of a homozygous RGMS two-type line, MS5001AB, was used to construct two suppression subtractive hybridization (SSH) libraries, from which differentially expressed genes were isolated and sequenced. Homology searches of the isolated sequences were performed using the NCBI BLAST server. Moreover, the sequences that produce homologs by BLAST were classified by MIPS of A. thaliana Genome Database according to the biological processes they participated in. Based on semi-quantitative RT-PCR of some differentially expressed genes, we selected two ESTs to obtain their full-length cDNA and genomic sequence by RACE technology. The gene sequences were analyzed using bioinformatics software. Finally, we constructed two plant expression vectors with sense and antisense ZeMYB35gene, which were introduced into tobacco by Agrobacterium-mediated transformation. Main results and conclusions are listed as follows:(1) Construction of subtractive cDNA libraries. Two subtracted cDNA libraries between fertile and male sterile plants, which contain1536clones, are constructed by suppression subtractive hybridization approach from disk florets. The subtraction efficiency of25-fold was obtained in the two libraries using the housekeeping gene β-tubulin as the reference. Blot hybridization against the1536clones was carried out to screen differentially expressed genes.525positive clones were obtained for sequenceing. The average lengths of fragments from the fertile and male sterile libraries were407bp and386bp, respectively. Finally,167nonredundant ESTs were obtained, among which 70were up-regulated in fertile buds, and97were up-regulated in male sterile ones. Results of homology searches showed that some genes from the fertile library were related to tapetum and/or microspore development, and some genes from the male sterile library were related to floret development or PCD. Semi-quantitative RT-PCR analysis showed that2of6genes were expressed only in young disk florets of fertile plants compared with that of male sterile plants. The other4genes were expressed only in leaves of male sterile plants. Fifty-nine ESTs from the fertile library are involved in carbohydrate metabolism, phosphate metabolism, and secondary metabolites. Eighty-one ESTs from the male sterile library are involved in protein synthesis, processing and degradation. These EST sequences will supply the foundation for further cloning of genes related to anther development. Further analysis of these genes will provide new insight into the molecular mechanisms underlying male sterile in Zinnia.(2) Cloning and sequence analysis of two differentially expressed genes related to floral development from Zinnia. Full-length of ZeMYB35and ZeSTR were cloned and their sequences were analyzed. The cDNA sequence of ZeMYB35is1143bp in length, with open reading frame (ORF) of885bp encoding294amino acids predicted by ORF Finder. ZeMYB35protein contains two typical MYB-like DNA-binding domains by Conserve domain program analysis, one of which is located in17th-62th amino acids, and the other in70th-113th amino acids. Nucleotide and amino acid sequence alignment analysis showed that ZeMYB35was homologous to MYB genes from other plants, with the highest homology (76%) with AtMYB35, which indicates that it is a member of MYB family, and belongs to the R2R3-MYB type. Genomic sequence of the gene was obtained by PCR, which is1264bp in length, consisting of three exons and two introns.The cDNA sequence of ZeSTR is1402bp, with ORF of1224bp, encoding407amino acids predicted by ORF Finder. The NCBI conserve domain program analysis found that the protein contains a STR-Synth structure domain, which is a typical structure feature of synthase, and is located in197th-284th amino acids. ZeSTR protein was homologous to various plant STR proteins, with the highest homology with Arabidopsis STR protein (AAO42227.1). The results showed that ZeSTR gene may code strictosidine synthase in Zinnia. Genomic sequences of the gene was obtained, with full-length of2 214bp, which contains four exons and three introns in the coding region. There are four replaced bases within the coding areas of ZeSTR cDNA and gDNA sequences, which may be caused by RNA editor. The results provide the foundation for further study of the structure and function of the gene.(3) Transformation of tobacco by ZeMYB35gene. To investigate the function of ZeMYB35gene, we constructed two plant expression vectors with sense and antisense ZeMYB35gene driven by CAMV35S promoter, which were then introduced into tobacco by Agrobacterium-mediated transformation. Stable transgenic plants were confirmed by PCR analysis, which showed that the purpose gene has been integrated into the genome of all the plants investigated. Compared with the untransformed tobacco plants, the pollen viability was slightly decreased in antisense transgenic plants. The growth of sense ZeMYB35transgenic tobacco plants was inhibited slightly. The result showed ZeMYB35gene may be related to pollen development.