Molecular Characterization Of RsFLC And RsSuSy In Radish(Raphanus Sativus L.)

Radish (Raphanus sativus L.) is an important Brassicaceae root vegetable crop in the world. China is the main place where radish was origined and domesticated, and has abundant germplasm. Radish is well accepted by the people in the world for its healthy value. Lots of cultivars of autumn, winter, spring and heat resistance types were released, and radish could be produced year-around.Late bolting cultivar development and the molecular mechanism of taproot formation are the important fields in radish genetic breeding.FLC (Flowering Locus C) is a key regulator of flowering time in vernalization required plants; SuSy (Sucrose synthase EC2.4.1.13) is a major enzyme of sucrose metabolism, involved in the formation of sink organs. We isolated the RsFLC and RsSuSy from early-/late-bolting lines and high-/low-ratio of roots lines, respectively. The gene structure, expression pattern, and the relationship between them and plant traits are also investigated. The results provide the evidence of revealing the molecular mechanism of flowering and flesh root expansion.The process of the floral bud differentiation could be divided into six stages, apical cone extension stage, flower primordium formation stage, sepal primordium polardium stage, stamen primordium polardium stage, pistil primordium polardium stage and petal primordium polardium stage. It can be happened on the tips of plant with two-three leaves at least. In addition, the corresponding vernalization requirement had closely relationship with the bolting characterization. It is suggested that vernalization requirement for floral bud differentiation in the two-three leaves stage was the best index for the bolting identification. Subsequently, the effect of this index was highly consistent with field plot in bolting identification.FLC genes were isolated from the early-and late-bolting radish inbred lines, named RsFLC1and RsFLC2, respectively, according to the homologous-cloning technique. The nucleotide sequence analysis showed that RsFLC was composing of7introns and6exons. There were MADS-box and K-box in the putative protein of RsFLC, was similar with other FLC in Brassica. RsFLC was highly expressed in the meristem of un-vernalization and late bolting radish line, but without in the root. The expression level of RsFLC can be reduced by vernaliztion, and more rapidly in the radish with early-bolting characterization and long exposuring to low temperature. Floral bud differentiation was initiated in the tips when RsFLC can not be detected was observed. It is suggested that the RsFLC could respond to vernalization which was correlated with the level of RsFLC transcription, and had a significant relationship with the status of flower bud differentiation.The alignment of RsFLC genomic DNA sequence, which was derived from15early-and late-bolting accessions, showed that there were two InDel regions, which was highly related with the bolting characterization. According to the two varied regions, two RsFLC gene specific markers were developed. The bolting characterization could be exactly identified with them, and also the genotype. These two gene specific marker would play an important role in radish late-bolting cultivar genetic development.Two isoforms of Sucrose Synthase genes, RsSuSy1(complete CDS) and RsSuSy6(partial CDS), were isolated from the inbred lines with the different ratios of root. The gene structure of RsSuSy1was not the same as AtSUS1, but similar in the domain of putative protein. There are two domains, Sucrose synth and Glycos_trasf1, in RsSuSy1, and Sucrose_synth also in RsSuSy6. It is revealed that RsSuSy1and RsSuSy6were belonged to the family of sucrose synthase, and related with sucrose metabolism. The expression patterns were also analyzed during the development of radish fleshy taproot. RsSuSy1was highly expressed in taproot, but the expression level of RsSuSy1-1, which was isolated from high ratio of root line was twice as RsSuSy1-2, the one was isolated from low ratio of root line. In contrast, RsSuSy6has a different expression pattern. RsSuSy6-2, driving from low ratio of root line, was expressed in the mature leaf at all the stages and in the phloem at expanding stage, whereas the expression of RsSuSy6-1, from high ratio of root line, was from the mature leaf to taproot along with the taproot development. It can be inferred that the transcript level of RsSuSy1significant positively corelated with the size of taproot, the sink organ of radish, and play an important role in the sucrose metabolism regulation and sink size; RsSuSy6may play an important for the leaf development and has no direct relationship with the taproot developing.