Effects Of TT8 And WD40 Transcription Factors On Anthocyanin Biosynthesis And Flowering Time In Brassica Oleracea L.

Anthocyanin is a kind of water soluble natural pigment which exists widely in plants,it belongs to flavonoid compounds.The expression of the structural genes controlling anthocyanins biosynthesis are mainly affected by MYB,b HLH and WD40 transcription factors and the complexes.Cabbage is one of the most important cruciferous crops,there are two common types of cabbage,purple cabbage and green cabbage.The purple cabbage can accumulate anthocyanins under natural conditions,but anthocyanins accumulation in green cabbage appear only under the lack of fertilizer,low temperature and other biological and abiotic stress conditions.BHLH,WD40 transcription factors had been found to be involved in anthocyanin biosynthesis,seed coat pigment accumulation in a large number of plants,however,there are few studies about the mechanism that how the two transcription factors control the synthesis of anthocyanin in green cabbage.In addition,it has not been reported whether bHLH and WD40 transcription factors could affect the plant flowering time.In this study,TT8 and WD40 transcription factors from cabbage were fused to a EAR-motif repression domain(SRDX)to get pCABarTT8 ER,pCABarWD40ER,pCABarTT8-WD40 ER vectors,then the vectors were transformed into cabbage.The functions of TT8 and WD40 transcription factors involved in anthocyanin accumulation and flowering regulation in green cabbage were explored.The main results in this study are as follows: 1.Obtained pCABarTT8 ER,pCABarWD40ER,pCABarTT8-WD40 ER transgenic cabbageThe pC ABarTT8 ER,pCABar WD40 ER,pC ABarTT8-WD40 ER expression vector were transformed into cabbage through agrobacterium mediated method.After several rounds of screening by added 8 mg/L phosphinothricin(PPT)in culture medium,we obtained the transgenic cabbage plants.And further PCR results of transgenic plants showed that the target gene had been successfully integrated into the genome of cabbage.2.The expression level of the genes related anthocyanin metabolism in leaves of pCABarWD40 ER,pCABarTT8ER,pCABarTT8-WD40 ER transgenic cabbageNo significant difference was observed of leaves anthocyanin accumulation of the pCABarTT8 ER,pCABar WD40 ER and WT plants.The expression of structural genes regulating anthocyanin biosynthesis were performed real-time PCR analysis in the leaves of pC ABarTT8 ER,pCABar WD40 ER and pCABarTT8-WD40 ER transgenic cabbage.The results showed the expression of DFR and ANR in pCABarWD40 ER plant leaves were significantly lower than WT,but only the ANR expression level was significantly lower than WT in pCABarTT8 ER cabbage.The color of pCABarTT8-WD40 ER leaves is lighter than WT,and the expression level of CHI,ANS,ANR and UFGT were significantly lower than WT.The results indicated that the function dominant inhibition of the TT8 and WD40 transcription factors alone would result decreased expression level for some anthocyanin biosynthesis structure genes in cabbage,and stronger inhibition effects would occur under the co-suppression of the TT8 and WD40 transcription factors.3.The seeds phenotype of pCABarTT8-WD40 ER transgenic cabbage and genes expression analysis related anthocyanin biosynthesisSeeds phenotype of pCABarTT8-WD40 ER after bud pollination 30,40 and 55 days were investigated.The seed coat color of the seeds after bud pollination 30 days was slightly weaker than wild type,but no difference of seed coat color of the seeds after bud pollination 40 and 55 days was observed compared with wild type.The real-time PCR analysis of the anthocyanin biosynthesis structure genes in three development seeds showed that the dominant inhibition of TT8-WD40 transcription factor did not affect the accumulation of seed coat pigment.4.The anthocyanin accumulation and anthocyanin biosynthesis structure genes expression in pCABarTT8-WD40 ER transgenic cabbage under low-phosphorus and low-temperature stressThe young leave,petiole,stem of wild type seedling show obvious anthocyanin accumulation but no anthocyanin accumulation in pCABarTT8-WD40 ER seedlings after the low-phosphorus stress of T1 generation pCABarTT8-WD40 ER seedling and wild type seedling.The expression level of DFR,ANS and UFGT genes in pCABarTT8-WD40 ER seedlings was significantly lower than wild type seedlings.The results indicated that the function dominant inhibition of TT8-WD40 transcription factors in pCABarTT8-WD40 ER under low-phosphorus stress result in expression level decreased of the downstream target genes that TT8-WD40 regulated,and the accumulation of anthocyanin decreased.In addition,the WT cabbage plants turned to purple under low-temperature stress,but both the pCABarTT8 ER,pCABarWD40ER plants almost no any color changed under the same stress condition.The results further suggested that the accumulation of cabbage anthocyanins in stress conditions requires the involvement of transcription factors TT8 and WD40.5.The gene expression analysis involves the flowering time in pCABarTT8ER?pCABarWD40ER?pCABarTT8-WD40 ER transgenic cabbage.Early flowering was observed of the PCABarTT8-WD40 ER transgenic cabbage in the field.The expression level of key vernalization gene VIN3 and promoting flower gene SOC1 in pCABarWD40 ER was significantly higher than wild type.In the pCABarTT8 ER transgenic plants,the expression level of the VIN3 was also significantly higher than wild type.The results indicated that the dominant inhibition of the TT8 and WD40 transcription factors had a certain promoting effect on flowering time in cabbage.Furthermore,we also found the co-dominant inhibition of TT8-WD40 function promoted the expression of the vernalization gene VIN3 and promoting flower gene SOC1 and inhibited the expression of late flowering gene FRI,caused early flowering of pCABarWD40-TT8 ER transgenic cabbage.