| As one of the most important cash crops,rape(Brassica napus)is the largest oil crop in China.At present,high-oil breeding is the main research direction of rapeseed breeding,and the improvement of rapeseed oil content has always been concerned.Anthocyanins,a kind of flavonoid compounds in plants which play an important role in the process of plants resisting biological and abiotic stresses.Now,healthy functional foods rich in anthocyanins are becoming more and more popular.Transcription regulation is an important regulatory factor in seed oil and anthocyanin accumulation.The b HLH(Basic Helix-Loop-Helix)transcription factor,GLABRA3(GL3)is known to regulate anthocyanin biosynthesis and trichome formation of Arabidopsis thaliana,but its function in seed oil accumulation is still unclear.In trichome formation,anthocyanin and oil accumulation,little is known about the regulatory functions of BnGL3-1,the homologous gene in B.napus.In order to analyze the regulatory effects of AtGL3 and BnGL3-1 on anthocyanin and oil accumulation and explore whether they have similar biological functions,this research used the A.thaliana gl3-3 and gl3-4 mutant,and the B.napus cultivar “ QINYOU Seven ” to preliminarily analyze their regulatory mechanisms and functional similarities and differences in anthocyanin and seed oil accumulation.The main results are listed as follows:(1)To comprehensively analyze the spatiotemporal expression pattern of AtGL3,the p AtGL3:GUS vector was constructed and transformed into the A.thaliana wild-type,and then the p AtGL3:GUS independent lines were used for GUS staining analysis,and meanwhile the expression of AtGL3 gene at different tissues and seed development stages was determined by quantitative reverse transcription PCR(q RT-PCR).The results showed that AtGL3 was widely distributed in vegetative and reproductive organs of A.thaliana,and it was highly expressed in young shoots and expanding true leaves during the initial stage of the transition from vegetative growth to reproductive growth,and there was also a large amount of the expression of AtGL3 in different stages of seed embryo development.Therefore,it is preliminarily speculated that AtGL3 may be involved in the growth and development of seedling and seed.(2)Based on GUS staining results and phenotypic differences in anthocyanin content and the number of trichomes in young shoots and expanding true leaves between wild-type and mutants,the tissue from the young shoots and expanding true leaves of A.thaliana wild-type and gl3-4 mutant after 20 days of germination were selected for the RNA-Seq experiment,and then the differentially expressed genes were bioinformatics analyzed to investigate GL3 downstream targeted genes at the genome-wide level in Arabidopsis young shoots and expanding true leaves.Then q RT-PCR was used to further verify the expression level of these potential target genes.This study has obtained several transcriptional factors and structural genes involved in anthocyanin accumulation and trichome development that acted and were significantly regulated downstream of AtGL3.(3)Through multiple sequence alignment and phylogenetic analysis of GL3 proteins from different plant species,it was found that BnGL3-1(QINYOU Seven)shared a high degree of sequence identity with AtGL3 from A.thaliana(84%)as well as homologues from rapeseed(Br GL3,98%;Bo GL3,99%)at the protein level.It is concluded that BnGL3-1 is closely related to AtGL3 and is conservative in evolution.When expressed in tobacco leaves as a fusion protein with green fluorescent protein,BnGL3-1 accumulated in the nucleus,consistent with it functioning as a transcription factor.(4)Ectopic expression of the BnGL3-1 gene in the A.thaliana gl3-3 mutant could restore the phenotype of reduced anthocyanin content and numbers of trichomes in Arabidopsis young shoots and expanding true leaves.At the same time,q RT-PCR results showed that many genes involved in anthocyanin biosynthesis and trichome formation were significantly regulated by BnGL3-1.This indicated that BnGL3-1 and AtGL3 have similar functions in regulating anthocyanin biosynthesis and and trichome formation.(5)The seed phenotypes of wild-type,mutants and gl3-3 35S:BnGL3-1-6HA transgenic plants were observed,and then the content of oil and anthocyanin in the mature seeds was measured,and the expression of genes related to important stages of seed development was detected.These results showed that BnGL3-1 and AtGL3 have similar biological functions in regulating lipid and anthocyanin accumulation in seeds.Both of them could promote anthocyanin biosynthesis and inhibit seed oil accumulation by regulating the expression of a series of genes related to seed anthocyanin and oil accumulation.In conclusion,the regulation mechanisms of BnGL3-1 and AtGL3 in anthocyanin and oil accumulation were systematically studied,and their similarities and differences were compared.This study showed that BnGL3-1 can be not only beneficial to improve the ability of B.napus to resist external adverse factors by regulating anthocyanin content and trichome density,but also contribute to the development of rapeseed stalk,a functional vegetable rich in anthocyanins by increasing the anthocyanin content in leaves and young stems at bolting stage.In addition,it also explored new gene resources for multi-functional utilization and high-oil breeding of Rapeseed by molecular breeding. |
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