Function Analysis Of The Key Gene BnaA10.FLC In Flowering Time Regulation Of Brassica Napus

Flowering is an important physiological phenomenon in the growth and reproduction of higher plants.Plants have evolved a complex and precise mechanism to control flowering time in response to endogenous signals and environmental changes.Vernalization is an important way for plants to respond to external environmental conditions and regulate the timely flowering of plants.FLC（FLOWERING LOCUS C）is a key gene in the vernalization pathway and encodes the MADS-box family of proteins and is an important flowering repressor.Rapeseed（Brassica napus L.）is an important oil crop in China.Brassica napus can be divided into three different ecotypes:spring type,semi-winter type and winter type according to their different requirement of vernalizayion.There are nine FLC paralogs have been mapped in B.napus,and Bna A10.FLC is an potential key gene that regulates the flowering time revealed by many QTL studies.The purpose of this study was to reveal the effects of different allelic variations on the function of Bna A10.FLC by analyzing the expression patterns of different haplotypes,so as to clarify its key role in the flowering decision of rapeseed.The main results are as follows:1.We found that the promoter of Bna A10.FLC in Darmor-bzh（winter type）had a MITE transposon insertion（named as haplotype I）and the promoter of Zhongshuang 11（hemi-winter type）had a 4.2 kb DNA transposon insertion（named as haplotype II）while the first exon of Bna A10.FLC in Westar（spring type）had a 7.6 kb retrotransposon insertion（named as haplotype III）.PCR amplification analysis of 130 varieties of B.napus futher revealed that the three haplotypes are ubiquitous.Additionlly,haplotype I was found only in winter types.2.q RT-PCR analysis found that the expressions of Bna A10.FLC in Tapidor and zhongshuang 11 were gradually decreased with the temperature drops from autumn to winter,but was always hardly decteced in Westar.Moreover,the expression level of Bna A10.FLC gene in Tapidor was highest in Zhongshuang 11 in the warm autumn environment.3.The subcellular localization analysis revealed that the Bna A10.FLC was localized in the nucleus which indicated that it could be a transcription factor as FLC in Arabidopsis.4.In spring rapeseed environment,BC₃F₁ lines containing the Tapidor Bna A10.FLC gene flowered later than recurrent parent Westar for 5 to 15 days.Meanwhile,three independent transgenetic lines（T₁）of Westar with Tapidor Bna A10.FLC gene had significantly higher expression level of Bna A10.FLC and flowered later than the negative control plants for about 30 days in the field of Wuhan.5.By mini35S::GUS alanlysis using the tobacco transient expression system,it was found that MITE structure has the function as an enhancer and the intermediate AT-rich fragment is the key modif for the function.The dual fluorescence reporter system also demonstrated that the MITE transposon indeed enhances the activity of the Bna A10.FLC gene promoter.In T₂ generation of transgenetic Arabidopsis,plants with Tapidor Bna A10.FLC plus promoter with MITE flowered significantly later than plants with same gene construction but without MITE.