Molecular Characterization Of Taproot Formation Traits In Radish(Raphanus Sativus L.)

Radish (Raphanus sativus L.) belongs to the Brassicaceae family and is an important worldwide vegetable crop with high nutrition and medical value. Fleshy tap root is the main edible part and its development is much related to the yield and quality in radish. The development of taproot is a complex morphogenetic process. The research of fleshy taproot development experiences a long period of morphological, physiological and biochemical description. However, the scarcity of knowledge on root development molecular mechanism contributes to difficulties in breeding, production, and storage. In the present study, the changes on proteome and the difference of gene expression during the taproot development of radish were analyzed by two-dimensional electrophoresis, differential display reverse transcription and cDNA library construction. The results would provide theoretical basis for studying the molecular characterization of taproot development and improving important horticultural traits in radish breeding program. Main research results were as follows.1. A proteomic approach was employed to study the mechanism of radish taproot development with the high advanced inbred line’NAU-ZQH’as material. Proteins were extracted from roots by using modified TCA-acetone-phenol extraction method and profiled by two-dimensional gel electrophoresis. The differentially expressed proteins were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A total of25protein spots displayed significant difference and17were successfully identified as either up-regulation (11spots) or down-regulation (6spots) in intensity relative to the development stages, of which,15had known functions. The identified proteins had functions related to sucrose and energy metabolism, protein metabolism and stability, resistance related proteins, signal transduction and regulation proteins. RT-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for17differential proteins. Of these proteins,11patterns of induced transcript accumulation were consistent with those of induced protein accumulation. 2. Differential-display reverse transcription-PCR (DDRT-PCR) was used to investigate the difference of gene expression during the taproot development of radish. A total of50differential fragments had been obtained in total of234selective amplifications.43fragments showed high sequence similarity to the genes of known or putative function, which were involved in transcriptional regulation, signal transduction, metabolism, transport channel and stress responding as well as protein metabolism such as NADH dehydrogenase, TIFY, translationally-controlled tumor protein, calcium-dependent protein kinase29,18S ribosomal RNA, aquaporin TIP2. The results indicated that the development of taproot was a complex process involving several pathways and stress responding.3. In this study, a full-length cDNA library from the taproot tissue was constructed and characterized. A total of10,052ESTs were generated from cDNA libraries of radish, of which were8,807high quality ESTs with an average length of515bp. Cluster analysis revealed the presence of1,510contigs and3,578singletons, representing5,088unigenes. Based on the sequence similarity search,4,684(92.1%),4,512(88.7%) and3,168(62.3%) of the unigenes showed homology with sequences in the Nr, Nt and SwissProt databases, respectively. According to molecular function of COG (Cluster of Orthologous Groups of proteins) classification,1,475unigenes were assigned and sorted into24groups. Based on the known or predicted annotation, the unigenes involved in general function prediction only ranked the first, followed by those in posttranslational modification, protein turnover, chaperones. A total of4,490unigenes were assigned to specific Kyoto Encyclopedia of Genes and Genomes pathways. The enzymes in the metabolism group were most represented and dominated by’carbohydrate’followed by’energy’metabolism.4. To investigate the profile of gene expression in radish taproot of different development stages, solexa sequencing was used to produce genome-wide gene expression profiling. The sequencing results showed that a large number of tags were generated from the taproot of two different stages, including254,922and145,414clean tags with more than one copy in the two libraries, respectively. Of these,53,941(21.16%) and35,435(24.37%) tags were matched to the reference genes. The tags with log2ratio>2or<-2(P<0.001) and FDR<0.01were characterized as the most differentially expressed genes and further analyzed, representing166up-regulated and292down-regulated genes, which were classified into17functional categories based on COG functional category. Moreover, the differentially expressed genes were queried against the KEGG pathway database and mapped to63KEGG pathways. Furthermore, the expression patterns of20genes selected were assessed by semi-quantitative RT-PCR, and the results showed general agreement with the Solexa data, which basically confirmed the reliability of our transcriptome analysis.5. To develop novel expressed sequence tags-simple sequence repeats (EST-SSR) markers in radish, a set of179SSRs, distributing in176unigene sequences, were detected by the software SSR Locator Ⅰ v.1, of which,50.84%were di-nucleotide, followed by tri-(29.61%), hexa-(9.49%), penta-(6.15%) and tetra-nucleotide (3.91%), respectively. GA/TC (25.69%) was the most frequent repeat type of all repeat types. Out of125primer pairs designed,110(88%) could generate unambiguous amplification products. Totally28EST-SSR primer pairs were selected for genetic diversity analysis in32radish genotypes. It was found that two to seven alleles could be detected with a mean of3.5and the polymorphism information content (PIC) values of these primers ranged from0.000to0.825with the average of0.500. The32accessions were generally divided into three main clusters at a similarity index value of0.60, which was mainly in accordance with the different biological characterizations of the accessions. In addition, the28EST-SSR primer pairs were further used to test the transferability on12accessions from three genera of Raphanus, Brassica and Arabidopsis in Brassicaceae. The results showed that18primer pairs (64.2%) could produce target PCR bands in the12accessions. The EST-SSR markers developed herein represented a valuable resource for the genetic diversity analysis, genetic mapping and marker-assisted selection in radish.6. An EST homolog to sucrose phosphate synthase (SPS) was found from the cDNA library, and the full length sequence of RsSPS1was obtained by homology cloning. The genome sequence was4,945bp including13extrons and12introns. The cDNA consisted of an open reading frame (ORF) of3,141bp and the predicted protein of1,044amino acids. The sequence of coding region showed the high homology of91%with SPS gene reported on Arabidopsis (GenBank accession No.AY039911). Phylogenetic tree analysis indicated that RsSPS1had a very close relationship with AtSPSl of Arabidopsis. Semi-quantitative reverse transcriptional-polymerase chain reaction and Real-time quantitative RT-PCR were performed to analyze the temporal and spatial expression pattern of RsSPS1. It was found that RsSPS1expressed in leaves, phloem and xylem of taproot with significantly different transcript level over the whole development stages.