Cloning And Functional Characterization Of Vernalization-related Gene BcFLC In Brassica Campestris Ssp. Chinensis

Plants in Brassica genus are a kind of vegetable crops with the highest yield and are widely cultivated throughout China. One of the major kinds of Brassica is Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis), most of which own a winter annual flowering habit. It is a habit that plants needs a period of cold (vernalization) to transfer from vegetative growth to reproductive growth, and to complete the life cycle. In practice, premature bolting is a serious problem not only causes a great of loss in production, but also reduces the commercial value. Anti-bolting breeding is the ultimate approach to solve this problem, which calls for the understanding of these species' flowering habits. Flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis) is a unique variety, which does not need vernalization to induce flowering, the so called summer annual flowering habit. It represents of an extreme early-bolting species, which can be produced all over the year in southern parts of China. Common Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis var. communis) has a similar genetic background with flowering Chinese cabbage, but it greatly differs from the latter in the requirement of vernalization. They provide us excellent materials in the study of vernalization in Brassica crops. Along with the development of molecular biological theories and technologies, the molecular mechanism of vernalization in Arabidopsis thaliana, which also belongs to Cruciferae, has been studied in-depth by many researchers. We could use the above resources for reference in our studies.By homologous cloning and rapid amplification of cDNA ends (RACE), vernalization-related gene BcFLC-1 and BcFLC-2 and their intron 1 sequences were already isolated from B. campestris ssp. chinensis var. communis cv. Shanghai-qing and B. campestris ssp. chinensis var. parachinensis cv. Sijiu, respectively. In order to fully understand the function of BcFLC and the causation of different requirement of vernalization between common Chinese cabbage-pak-choi and flowering Chinese cabbage, we cloned the promoter of BcFLC, and constructed plant expressing plasmid vectors of anti-sense RNA and over-expression vectors using fragments of BcFLC. By infecting common Chinese cabbage-pak-choi and flowering Chinese cabbage through Agrobacterium-medmted transformation, respectively. the transgenic plants of common Chinese cabbage-pak-choi and flowering Chinese cabbage were obtained consequently. And the flowering habits of the transgenic plants were observed subsequently. The results provided available information on exploring the function of BcFLC and the mechanism of vernalization in Brassica crops. The results are as follows:(1) The nucleotide sequences of the coding areas of BcFLC-1 and BcFLC-2 were analyzed by bioinformatics software, the results of which showed a conformity of 99.34%. MADS-box and K-box, which were closely related to flowering in Arabidopsis, existed in the same location of the both amino acids sequences. Only one amino acid difference was found outside the conservative regions. The two intron 1 sequences showed a similarity of 91.67%, while the two promoter sequences got a 97.1% consistency All these indicated that BcFLC-1 and BcFLC-2 had no substantial difference.(2) The over-expression and anti-sense RNA expressing plasmid vectors with CaMV 35S promoter, pBI35S-BcFLC1 and pBI35S-BcFLC2 were constructed using fragments of BcFLC. Molecular identification showed that both of the vectors along with the pBI35S (negative control) were introduced into Agrobacterium tumefaciens strain LBA4404.(3) 230 Kan^R plantlets of 48 regenerated lines were obtained on the efficient genetic transformation system based on Yu XL et al (2002). There were 20 regenerated lines of pBI35S-BcFLC1 plantlets with an efficiency of 3.608% and 2 regenerated lines of pB1121 plantlets with an efficiency of 1.831% in flowering Chinese cabbage. Similar situation happened in common Chinese cabbage-pak, while there were 18 regenerated lines of pB135S-BcFLC2 plantlets with an efficiency of 2.272% and 2 regenerated linos of pB1121 plantlets with an efficiency of 1.210%. The frequency of positive Kan^R seedlings is 84 78% examined by PCR and Southern blot.(4) Flower buds in transgenic flowering Chinese cabbage showed 54 days after differentiating culture or when the 10^th leaf came out, while the control showed flower buds 45 days after differentiating culture or when the 8^th leaf came out. Transgenic common Chinese cabbage-pak-choi displayed great difference with the control, by showing flower buds without vernalization on an average of 78 days after differentiating culture of when the 10^th leaf came out.