Preliminary Study On The Molecular Mechanism Of Continuous Vernalization Propagation Causing Winterness Weakened On Chinese Cabbage

Chinese cabbage〔Brassica campestris L.ssp. pekinensi（sLour）Olsson〕is originated in China,it is one of the most important vegetable crops of Brassica species. It has the phenomenon ofpremature bolting that bolting time occurs before its vegetative plants ripen, and causes asignificant reduction in economic benefits because of losing edibility. In spring and summer,cabbages are easily premature bolting in high altitude area of china. Spring Chinese cabbage iseasily occurred premature bolting in the process of production in the northern area of China. Inrecent years, cultivating strong winterness and bolting-resistant varieties had become the primarytask of breeding research.At present, the phenomenon of winterness weakened exists in the breeding throughcontinuous vernalization. There are studies reported that after vernalization, methylation levelreduced and easy to bolting and flowering. Therefore, in order to indicate the mechanism ofweakened winterness of offspring under continuous vernalization in molecular level and guidancefine variety breeding of Chinese cabbage breeding, we used MSAP (methylation sensitiveamplification polymorphism) to detect genomic methylation level. Meanwhile, we detected theexpression of gene FLC1, FLC2, FCA and SAMS through semi-quantitative RT-PCR(semi-quantitative reverse transcription-polymerase chain reaction), and detected the expression ofgene SAMS, FLC1through real-time RT-qPCR (real time fluorescence quantitative RT-PCR) indifferent treatments of Chinese cabbage. The maximum difference of expression quantity wasbetween the stock plant collecting seeding and the plant of vernalization three generation。Finallyaimed at the different expression of FLC1, we chose above both to make BSP (bisulfite sequencingPCR) analysis to analyze how promoter region methylation affected gene FLC1expression. Mainresults are as follows:(1) Experimental materials were plants of stock plant collecting seeding, vernalization onegeneration, vernalization two generation and vernalization three generation of A161and A54-1inbred line. Through detecting their genomic DNA methylation we found that the genomic DNAmethylation level was reduced with the vernalizaiton generation increasing in both cabbagevarieties. (2) SAMS gene was reduced with the vernalization generation increasing in A161inbred linethrough semi-quantitative RT-PCR and real-time RT-qPCR detecting. Gene SAMS is the onlygenerate path of SAM, which is the important methyl donor. Due to this we concluded themethylation level was reduced with the vernalization generation increasing, which was accordingwith the result of MSAP detection.(3) Through semi-quantitative RT-PCR detecting the expression of FLC1, FLC2and FCA, wefound the expression of FLC1and FLC2was reduced as the vernalization generation increasingand the expression of FCA was increased as the vernalization generation increasing. This resultindicated the winterness of plants were weakened as the vernalization generation increasing, andwas according with the result of field investigation. The result of FLC1expression throughsemi-quantitative RT-PCR and real-time RT-qPCR detecting had same conclusion.(4) Through detecting the methylation of FLC1promoter region by BSP (bisulfite sequencingPCR) analysis, we found the C of-293bp was demethylated in the plant of vernalization threegeneration of A161inbred line. This C methylation in the promoter region of FLC1mightparticipate in the change of expression quantity of gene FLC1.