Research On The Transcriptional Regulation Mechanism Of BoPIF4 And BobHLH66 Transcription Factors On Key Genes Of Postharvest Broccoli Yellowing

Broccoli(Brassica oleracea L.var.italic)is rich in flavonoids,ascorbic acid(VC),glucosinolates,sulforaphane and other nutrients and bioactive substances,which attracte the attention of consumers.In recent years,the cultivation area has been expanding.However,broccoli is not resistant to storage and transportation.After harvesting,the flower ball is easy to turn yellow,accompanied by changes in nutrients,which seriously affects the quality and commercial value of broccoli.Therefore,it is of great theoretical and practical significance to study the yellowing mechanism and explore the regulation technology.In this paper,the broccoli of the Naihan-Youxiu was used as the test material.Firstly,the yellowing rule of broccoli under different temperatures was analyzed.Then the transcriptome sequencing technology was used to study the differential expression of genes during broccoli yellowing,and the function of differentially expressed genes(DEGs)and the metabolic pathways involved in them were analyzed.On this basis,the key enzyme genes of pigment metabolism in broccoli yellowing process were further screened,and the transcriptional regulation pattern of transcription factors on key genes was studied.The mechanism of revealing post-harvest yellowing of broccoli at the molecular level was based on the above research contents.The conclusions of this paper are as follows:(1)At the temperature set in this test(20?,10? and 0?),the higher the temperature,the earlier the broccoli turned yellow,the more serious the degree.During the process of yellowing,the a* and b* values of broccoli increased,the h° value decreased;the shape of buds did not change significantly,and the buds did not bloom;The yellowing began from the base of the buds,accompanied by this process,the surface tissue damage of buds,chloroplast transformation to chromoplasts,chlorophyll development abnormalities.The content of total chlorophyll and chlorophyll a in broccli buds decreased,the chlorophyll b content decreased,and the carotenoid content increased.Color intensity correlated significantly with the chlorophyll b,?-cryptoxanthin,and ?-carotene contents,which were associated with increased yellowing of plant tissues.(2)The first,5th and 12 th day were the slight yellowing and serious symptoms stages of broccoli after storage at 10?.Sampling at 0 d,5 d and 12 d,respectively,transcriptome sequencing analysis,screening 1717 differentially expressed genes(DEGs),DEGs enriched into 92 metabolic pathways.Transcriptome sequencing profiled 6,5,and 4 differentially expressed genes(DEG)involved in chlorophyll metabolism,carotenoid biosynthesis,and flavonoid biosynthesis,respectively.There were 579 differentially expressed transcription factors.The transcription factor gene ontology(GO)categories showed that the MYB,b HLH,and b Zip gene families were involved in chlorophyll metabolism.In addition,the transcription factor families included NACs and ethylene response factors(ERFs)that regulated carotenoid biosynthesis.The flavonoid biosynthetic pathway was mainly regulated by MYB,NAC,WRKYs,MADS,and b Zip.Reverse transcription polymerase chain reaction further confirmed that Bob HLH66,Bo PIF4,Bo LOB13,Bo NAC92,and Bo APL were vital transcription factors that potentially regulated the pigments metabolism.During the yellowing process,Fv/Fm and chlorophyll content decreased,non-photochemical quenching(NPQ)levels and carotenoid content increased.(3)The promoters of Bo CAO and Bo HYD genes and the full-length coding region of Bo PIF4 and Bob HLH66 genes were successfully cloned from broccoli.Bo PIF4 and Bob HLH66 were nuclear localization proteins and were identified as members of the b HLH transcription factor family.Bo PIF4 and Bob HLH66 positively activated the chlorophyll-degrading key enzyme genes(Bo CAO)and the carotenoid biosynthesis key enzyme genes(Bo HYD)by specific binding to G-box and GCACGTGC elements,respectively.Among them,the transcriptional activation ability of Bo PIF4 was significantly higher than that of Bob HLH66.(4)The full-length fragments of the coding region of Bo PIF4 and Bob HLH66 homologs genes(At PIF4 and Atb HLH66)were cloned from Arabidopsis thaliana.Then,the target gene fragments were successfully recombined into overexpression and gene interference vectors,respectively.Overexpression of At PIF4 and Atb HLH66 increased the transcriptional levels of At CAO and At HYD genes,and further induced the decrease of chlorophyll content and carotenoid content in Arabidopsis,which aggravated the abnormal development of chloroplasts and promoted the leaves of Arabidopsis thaliana yellowing.Conversely,the transcription levels of At CAO and At HYD in the RNAi lines were reduced,and the role in protecting chlorophyll degradation,inhibiting carotenoid synthesis,and maintaining chlorophyll structure was significant,and the RNAi transgenic plants exhibited a green phenotype.(5)The melatonin-treated broccoli appeared to have a yellowing phenotype delayed by 4 days compared to the control at 20?.Melatonin treatment inhibited the degradation of chlorophyll and the accumulation of carotenoids in broccoli by inhibiting the transcriptional levels of key genes(Bo CAO and Bo HYD)and transcription factor encoding genes(Bo PIF4 and Bob HLH66),thereby delaying the yellowing of broccoli.At the same time,the treatment effectively increased the content of flavonoids and VC.