Effects Of Exogenous Cabbage Genes On Flowering And Related Gene Expression In Chinese Cabbage-Cabbage Translocation Line ’19a-10’

Chinese cabbage is originated in China,which is rich in nutrition and has the reputation of the classic vegetables’.Premature bolting and flowering will lead to the decrease of the quality and quantity of vegetative organs in Chinese cabbage.In this study,the progenies of microspore translocation lines with cabbage chromosome 2 fragment(containing cabbage BoFLC3 gene)were generated by selfing,and the materials with and without exogenous cabbage fragment translocation lines were obtained by molecular marker identification,transcriptome sequen cing and real-time PCR analysis were performed.The main results are as follows:1.The progenies of two different Chinese cabbage microspore translocation lines which added with ragment of cabbage chromosome 2 were identified by the molecular marker of specific regions of BoFLC3 gene.The proportion of plants which contains BoFLC3 marker within ’19A-10’ lines was 69.6%.and the ’19A-22’ lines was 51%.While the retention ratio of the next inbred generation of ’19A-10’ lines was 67.6%.2.8 InDel markers:C03-1,C03-2,C03-39,C03-4,C03-40,C03-42,C03-44 and C0345(BoFLC3 gene position is between C03-40 and C03-44)of C03 linkage group were used to the test the first selfing generation plants of ’19A-10’lines,only 4 InDel markers between C03-4(physical position 1613774 bp)and C03-44(physical position 3747840 bp)amplified bands consistent with cabbage in chinese cabbage translocation line(named’M’),and confirmed that the inserted chromosome fragment of cabbage was about 2.08Mb;The retention ratio of cabbage markers C03-4,C03-40,C03-42 and C03-44 in the first selfing generation plants of translocation lines ’M’ was about 97%,while C03-1,C03-2,C03-39 and C03-45 were completely lost in the first selftng generation plants of’19A-10’ lines.3.The selfing progeny of translocation lines seeds were treated with low temperature for 0,1,2 and 4 weeks respectively,and sowed at the same time.The budding and flowering time of translocation lines ’M’ containing exogenous cabbage fragments and translocation lines’N’ without exogenous cabbage fragments was investigated.The results showed that the two materials treated at low temperature for 0 and 1 week did not bloom;After 2 weeks of low temperature treatment,the budding and flowering time of’M’ was later than ’N’ and there was significant difference in flowering phenotype between the two materials;After 4 weeks of low temp erature treatment,the budding and flowering time of’M’ was 30 days and 38 days respectively,while as for ’N’ was 28.3 days and 35 days respectively,which indicated that flowering time of Chinese cabbage translocation lines was affected by the insertion of exogenous cabbage fragments.4.Transcriptome sequencing was carried out on leaf tissues of translocation lines ’M’and ’N’,both ’M’ an d ’N’ seeds were vernalized for 0,1 and 4 weeks,’N’ set as con trol group and ’M’ set as experimental group.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses and Gene ontology(GO)were subsequently conducted,29 DEGs related to flowering were found during the transition from vegetative growth to reproductive growth,including 3 up-regulated DEGs and 26 down-regulated DEGs.83%of those down-regulated DEGs participated in the regulation of flowering through photoperiod pathway,which were CO,HY5,SPA3,PAT1,CK2a,LHY1,CCA1 and CCA1.In addition,also identified some DEGS related to flower development,such as ERF107 and DEAR2.There are 4 differentially expressed genes related to flowering in Chinese cabbage translocation lines,which are FLC3,FY,AGL2 and MYB56.5.RT-qPCR was performed on differentially expressed flowering genes FLC3,FY and MYB56 which also existed in exogenous cabbage fragments.The relative expression level of FLC3 gene was signifi cantly lower in ’N’ compared to ’M’ after 0 and 1 week seeds vernalization treatment;The relative expression level of FY gene in’M’ always higher than’N’ in every sampling point of time.While the relative expression level of MYB56 in’M’ was significantly lower than ’N’ at 3 sampling point of time.This result indicates that FY inhibits the expression of FLC3,and the expression level of MYB56 is consistent with the plant phenotype.In a word,FLC3,FT and MYB56 genes expression level trend is consistent with what we found about those 3 genes in transcriptome data.Furthermore,the spatio-temporal expression patterns of FLCs homologous genes in the 5th and 7th leaves of’M’ and ’N’ first selfing progenies generation shows that there was no significant difference in relative expression level between the 5th and 7th leaves,which further verified the reliability of transcriptome data and the accuracy of sampling parts.