Study On Vernalization-related Genes In Brassica Campestris Ssp. Chinensis L.var. Utilis Tsen Et Lee (Purple Flowering Stalk) And Molecular Mechanism Of Its Flowering Early

Purple Flowering Stalk (Brassica campestris ssp. chinensis L.var. utilis Tsen et Lee) is one of the most popular vegetable crops in the south of China, and has been cultivated for thousands of years. Unlike other Brassica species, such as Chinese cabbage which is a vernalization-responsive plant, Purple Flowering Stalk can flower early without vernalization. Purple Flowering Stalk which is a unique variety of Chinese cabbage has similar physiology character and genetic background with Chinese cabbage. But they greatly differ from requirement of vernalization and flowering time. Purple Flowering Stalk has a weak requirement of vernalization and flowering early. After reviewing the advances and achievements in research on physiology character and molecular biology of Purple Flowering Stalk which is a natural early flowering mutant, this research addressed the FLC gene which regulates flowering of plant. FLOWERING LOCUS C (FLC), which encodes a MADS-box transcription factor acts as a repressor of the floral transition. Weak expression of FLC and its functional deletion results in early flowering of Arabidopsis and Chinese cabbage et al. which have vernalization responsive. We investigated the molecular mechanism of Purple Flowering Stalk flowering earlier and FLC homologous gene, BrpFLC, and other vernalization-related genes from Purple Flowering Stalk and obtained the main results as follows:(1) Two alternative splicing patterns, one with different parts of intron 6 retained and one with the entire exon 6 excluded from the transcript, were identified in Purple Flowering Stalk. These two alternative splicing patterns named BrpFLC1-1 and BrpFLC1-2 were confered having no function(2) Another FLC homologous genes cloned from Purple Flowering Stalk were named as BrpFLC2, BrpFLC3 respectively. They contain 91 bp, 618 bp ORF which encode 196, 197 amino-acid sequence with molecular mass of 21.91, 21.67kD and isoelectric point of 8.84, 9.08, respectively. According to the blast results in GenBank database ,the cDNA nucleotide sequence shares 84%, 82% identity with AtFLC(NP₁96576)of Arabidopsis . BrpFLCs of Purple Flowering Stalk have very high identity with that of Chinese cabbage.(3) Bioinformatics prediction on BrpFLC genes nucleotide and encoded amino acid residue sequence indicates that the encoded proteins are MADS-box transcription factor and possibly have multi phosphorylation sites, but no 3 O-glycosylation sites. These proteins have trans-membrane structures which were predicted in FLC of Arabidopsis and Chinese cabbage. BrpFLCs have similar secondary structure with most of MADS-box transcription factor. They may regulate expression of genes as transcription factor locate in a nucleolus.(4)Study of expression pattern of BrpFLC indicated that BrpFLC1,BrpFLC2 were found to be ubiquitously expressed in roots, stems, leaves and cotyledon of purple stem mustard. BrpFLC3 was found to be expressed highly in leaves and stems, leaves and cotyledon but low in roots. Reduced BrpFLC1,BrpFLC2 and BrpFLC3 expression were observed in Purple Flowering Stalk which was vernalized. The expression of BrpSOC1and BrpVIN3 increased after being vernalized, but there was no obviously change of expression of BrpFRI.(5) Study of flowering time of Purple Flowering Stalk indicated that Purple Flowering Stalk which flowers much earlier than Chinese cabbage was a natural early flowering mutant. Its transfer from vegetative growth to reproductive growth did not need vernalization, but it has vernalization responsive. Vernalization decreased the flowering time of Purple Flowering Stalk.(6)Three BrFLC1 alleles with alternative splicing patterns, including two with different parts of intron 6 retained and one with the entire exon 6 excluded from the transcript, were identified in addition to alleles with normal splicing. The analysis conferred that a G/A polymorphism at the 5'splice site in intron 6 of BrFLC1 is associated with splicing mutation in the BrFLC1 gene and flowering-time variation in Purple Flowering Stalk.(7) Upstream flanking region of 5'UTR of BrpFLC2 is similar with that of BrFLC2 in Chinese cabbage. It contains several TATA-box, CAAT-box and other cis-acting elements. Intron 1 of BrpFLC1 and BrpFLC2 are similar with that of BrFLC1 and BrFLC2 in Chinese cabbage. We also detected the sequence around initiation codon of BrpFRI which is a most common mutation site inducing early flowering in Arabidopsis. None of these regions we detected caused Purple Flowering Stalk flowering early.(8) Comparing with Chinese cabbage, BrpFLC1,BrpFLC2 and BrpFLC3 have weak expression in Purple Flowering Stalk. The weak expression of BrpFLC may cause Purple Flowering Stalk flowering early to some extent.(9)A fragment of VIN3 homologous gene which contains the PHD and FNIII region cloned from Purple Flowering Stalk was named as BrpVIN3. According to siRNA sequence design principles, a RNAi expression vector of targeting gene BrpVIN3 was constructed and will be transformed into Chinese cabbage. This research provides the foundation for further study on the function of VIN3 in Brassica species.