Anatomy Observation, Gene Cloning And Characteristics Analysis In Relation To Floral Development Of Ziziphus Jujube Mill

Chinese jujube (Ziziphus jujube Mill.), one member of the Rhamnaceae, is originated from China and has more than4000-year history of cultivation. It is an economically important fruit tree species in China, well-known for its fruits with high dietary value and medical value. In contrast to other common fruit trees such as apple, pear, and citrus, the Z. jujube exhibits unique flowering characteristics. For example, floral buds geminate on the fruit-bearing shoots, complete differentiation and develop into mature fruits along with the fruit-bearing shoots. In particular, the tree has a short juvenile phase preceding the production of flowers and fruits in the reproductive phase. It can blossom and produce fruits in the same year the seedling is planted. Therefore, elucidation of flowering mechanism of Z. jujube is of great significance to shortening and adjusting the juvenile phase, genetical modification, and new cultivars breeding. In this paper, systematic studies were conducted on the development of Z. jujube floral organs and related expressed genes via comparative anatomy experiment, SSH libraries construction, cloning, expression pattern and function detection of floral organs-related genes etc. The main results are as follows:1. Studies on comparative anatomy of Z. jujube floral organs. The development of Z. jujube ’jinsi No.4’ flower bud differentiation was divided into six stages:predifferentiation, fiffertiation phase, sepal formation, petal formation, stamen formation and pistil maturation.Z. jujube ’jinsi No.4’ was four ventricles in each anther; anther wall was4to5layers; there were the epidermis,1-2layers endothecium, middle and glandular tapetum layers; the development of the anther wall was basic type. The meiosis in the microspore mother cells were belonged to a simultaneous type and the microspores were arranged in a tetrahedron shape in the tetrads; the shape of pollen was a triangular germination vale with three germinal furrow; The ripened pollen was of2-cell type. Megaspore mother cell meiosis to the formation of four spores were linear arrangement. After three mitosis, the formation of mature embryo sac was eight-nucleate with seven cells. Anatropous ovule, bitegmic, crassinucellar and a Polygonum type. Between the internal anatomic structure and flower morphology were stably relationship in the developmental process of Z. jujube ’jinsi No.4’.2. Construction of Suppression subtractive hybridization libraries. Using developing floral buds and leaves during different stages as materials, total RNA extraction, mRNA prufication, cDNA synthesis, digestions, and ligations were carried out in turn. The cDNAs from floral buds and leaves were used as Tester and Driver respectively, two SSH libraries were constructed via the method of suppression subtractive hybridization. About3578clones were obtained from the SSH library of Z. jujube floral buds, of which3294were positbve, the recombination frequency was92.05%. About4055 clones were obtained from the SSH library of Z. jujube floral buds, of which3660were positbve, the recombination frequency was90.26%. Positive clones were selected at random and assayed by PCR with primers T7/SP6. It was found that above95%of clones from both two SSH libraries carried insertions. The size of the insertions ranged from200bp to2000bp, of which most focused on approximate1000bp, suggesting the insertions from two SSH libraries met the requirements in next work.3. Informatics analysis of EST sequences of Z. jujube floral organs.1000positive clones were selected at random and sequenced.912original sequences were obtained, of which883were high-quality ESTs with the size being above100bp. Among these,598ESTs were distributed from1101bp to1300bp.96unigenes were obtained by sequence assembling that included43contigs and53Singletons.73ORFs (open reading frame) were predicted from96Unigenes. Using EST sequences of96Unigenes as queries for Blast analysis in Nucleotide Database,72could search out homologous sequences. Among these homologous sequences, two floral development related genes, i.e.MADS7(GenBank accession No. EF440352.1) of Prunus persica and MADS1of Pinus radiata (GenBank accession No. U42399.1) were found. Blast analysis in Non-redundant Database revealed that70Unigenes could search out homologous sequences, of which two floral development related genes, i.e. SEPALLATA1of Citrus unshiu and MADS-box of Pinus radiata were found. Blast analysis in SWISSPROT revealed that43Unigenes could search out homologous sequences. Blast analysis in COG database showed that20annotated sequences were obtained and were classified into7groups, i.e., Translation, ribosomal structure and biogenesis, Posttranslational modification, protein turnover, chaperones, Carbohydrate transport and metabolism, Lipid transport and metabolism, Replication, recombination and repair, Cytoskeleton, and Protein binding. The numbers of the annotated sequences were5,4,4,3,2,1, and1, respectively.4. Informatics analysis of EST sequences of Z. jujube leaves.1000positive clones were selected at random and sequenced.806original sequences were obtained, of which735were high-quality ESTs with the size being above100bp. Among these,438ESTs were distributed from1101bp to1200bp.86unigenes were obtained by sequence assembling that included45contigs and41Singletons.70ORFs (open reading frame) were predicted from86Unigenes. Using EST sequences of86Unigenes as queries for Blast analysis in Nucleotide Database,71could search out homologous sequences. Blast analysis in Non-redundant Database revealed that66Unigenes could search out homologous sequences. Blast analysis in SWISSPROT revealed that41Unigenes could search out homologous sequences. Blast analysis in COG database showed that23annotated sequences were obtained and were classified into6groups, i.e., Carbohydrate transport and metabolism, Amino acid transport and metabolism, Coenzyme transport and metabolism, Energy production and conversion, Replication, recombination and repair, Celluar component. The numbers of the annotated sequences were12,3,2,2,2, and2, respectively.5. Cloning, sequence analysis and expression patterns of ZjMADS1-box. Based on known476bp EST sequence for ZjMADS1-box gene, specific primers were designed for3’RACE and5’RACE PCRs. Accoringly, the full-length cDNA of ZjMADSl-box were obtained by assembling3’and5’ teminal sequences. The full-length cDNA of ZjMADSl-box was957bp with5’non-transcription region of70bp,3’non-transcription region of224bp and complete CDS of663bp, encoding220amino acids. ZjMADSl-box exhibited characteristic structual of B type, MADS-box, ie., contained MADS domain, K domain, and PI domain. At nucleotide and amino acid level, the ZjMADSl-box showed the lowest similarity with CaPI (69.7%,61.8%), and the highest with MqPI (81.3%,72.6%). The predicted molecular weight and isoelectric points of ZjMADS1-box were25587.5Da and9.75, respectively, and belonged to unstable protein. Hydrophobicity index of the ZjMADSl-box varied from-3.033to2.100, exhibiting hydrophilicity in general. The ZjMADSl-box did not contain signal peptide. Secondary structure prediction showed that the ZjMADS1-box contained28Helixs,27Strands, and31Coils. The ZjMADS1-box gene was not expressed in vegetative organs, but in floral buds.6. Cloning and informatics analysis of ZjSEPl. Based on known338bp EST sequence for ZjSEPl gene, specific primers were designed for3’RACE and5’RACE PCRs. Accoringly, the full-length cDNA of ZjSEPl were obtained by assembling3’and5’teminal sequences. The full-length cDNA of ZjSEPl was1149bp with5’non-transcription region of106bp,3’non-transcription region of308bp and complete CDS of735bp, encoding244amino acids. ZjSEP1exhibited characteristic structual of E type, MADS-box, ie., contained MADS domain, K domain, and SEP domain. At DNA sequence level,the ZjSEP1showed the lowest similarity with LeMADS1(66.8%), the highest with PpMADS7(84.2%). At amino acid sequence level, the ZjSEP1showed the lowest similarity with CaMADS1(61%), the highest with PpMADS7(86.1%). The predicted molecular weight and isoelectric points of ZjSEP1were28073.9Da and9.13, respectively, and belonged to unstable protein. Hydrophobicity index of the ZjSEPl varied from-2.322to2.122, exhibiting hydrophilicity in general. The ZjSEPl did not contain signal peptide. Secondary structure prediction showed that the ZjSEP1contained27Helixs,26Strands, and32Coils. In The ZjSEP1gene was not only expressed in vegetative organs including fruit-bearing shoots and axillary buds, but also in reproductive organs, i.e. floral buds in different developing stages.7. Cloning and informatics analysis of ZjLFY. A446bp cDNA was amplified by degenerate RT-PCR from floral buds of Z. jujube and was identified as LFY homologous gene by Blastn analysis. Based on the known sequence, the full-length cDNA of ZjLFY were cloned via RACE technique. The ZjLFY sequences were subbmited to GenBank and the Accession No. was JN165097. The full-length cDNA of ZjLFY was1517bp with5’non-transcription region of50bp,3’non-transcription region of258bp and complete CDS of1209bp, encoding402amino acids. The amino acid sequence of ZjLFY contained two viable regions and two conserved regions. At DNA sequence level, the ZjLFY showed the lowest similarity with OiLFY and S1LFY (60.7%), the highest with CmLFY (77.2%). At amino acid sequence level, the ZjLFY showed the lowest similarity with OiLFY (57%), the highest with JrLFY (83%). The predicted molecular weight and isoelectric points of ZjLFY were45221.7Da and6.29, respectively, and belonged to stable protein. Hydrophobicity index of the ZjLFY varied from-3.667to1.756, exhibiting hydrophilicity in general. The ZjLFY did not contain signal peptide. Secondary structure prediction showed that the ZjLFY contained36Helixs,40Strands, and47Coils. 8. Expression pattern analysis of ZjLFY. Zjactin gene of Z. jujube was isolated by RT-PCR and used as a reference gene for expression analysis of ZjLFY gene. Semiquantitative RT-PCR analysis revealed that ZjLFY gene was expressed in both reproductive organs and vegetative organs. In general, the ZjLFY gene was expressed at all stages of floral bud development, showing high expression level. But the expression level decreased with floral buds approaching to maturity. A slight transcript level of ZjLFY was also detected in different vegetative organs including fruit-bearing shoots, vegetative shoots, stems, mature leaves, and axillary buds, whereas ZjLFY mRNA accumulation was higher in fruit-bearing shoots than in the other four organs. The profile of ZjLFY express suggested that expression levels of ZjLFY fluctuated in different organs with higher expression level in reproductive organs than in vegetative organs.9. Function analysis of ZjLFY gene by transformed into Arabidopsis. ZjLFY cDNA and pBI121vector were double-enzyme cleavaged, extracted, ligated, and recombined as recombinant pBI-ZjLFY that was whereafter transformed into Agrobacterium tumefaciens LBA4404. By using pollen-tube pathway, the pBI-ZjLFY was transformed into10wild-type Arabidopsis. TO generation seeds were collected and cultured as positive seedlings by resistance screening. T1generation transgenic Arabidopsis seedlings were screened by PCR assay. Then seeds were collected by self-pollination and T2generation transgenic homozygous arabidopsis plants were screened and cultured accordingly. It was found that T2generation plants flowered earlier than the wild-type plants by11days.