Cloning And Functional Characterisations Of Genes Orange Flower In Brassica Napus

Brassica napus is a valuable crop used as a source of edible plant oil,vegetable,fodder,biofuel and nectar.Along with the success of the rapeseed flower tourism in different regions in China,the cultivation of rapeseed has brought huge economic benefits to the ornamental tourism industry,and the ornamental value of B.napus has gradually attracted the attention of researchers in rapeseed genetics and breeding.Cloning and functional analysis of the key genes that control rapeseed flower color will provide a theoretical basis for revealing the molecular mechanism of rapeseed flower color formation,and provide a theoretical basis for creating colorful rapeseed germplasm resources by using bioengineering technology.In this study,by using map-based cloning approach,two genes controlling flower color phenotype,BnaC09g07550D and BnaA09g07610D,were isolated from segregating populations constructed from the orange-flowered mutant O271 and the yellow-flowered B.napus line Y409.HPLC,qRT-PCR,transgenic complementary test,CRISPR/Cas9,subcellular localization and transcriptome sequencing techniques were used in this study to study the function of these two genes.The main results are as follows:1.Genetic studies showed that the orange-flowered phenotype was controlled by two recessive nuclear genes（of1 and of2）.2.The HPLC analysis of the petals of parents and BC₃F₁ yellow-flowered plants at different development stages confirmed that the color change from yellow to orange were primarily due to the significantly enhanced accumulation of lutein and sharp decline in the concentrations of violaxanthins.3.Measurements of the endogenous ABA levels were performed in petals and leaves of yellow-and orange-flowered plants.The ABA level in leaves and petals showed no significant differences between the orange and yellow-flowered plants.4.Using two BC₃F₁ populations,OF1 was further narrowed down to a 25-kb genomic region on the C09 chromosome,and OF2 was pinpointed locus to a 196-kb region a 196 kb region1 on the A09 chromosome.Analysis of the gene annotation information in these two regions revealed that there was a homologous gene of Arabidopsis zeaxanthin cyclooxygenase in both regions,which was named as BnaC09.ZEP and BnaA09.ZEP,respectively.The results of RT-PCR and qPCR showed that these two genes are normally expressed in yellow petals,but not in orange petals.Comparative sequencing results showed that the orange-flowered mutant lost BnaC09.ZEP,and a fragment of 1695bp in BnaA09.ZEP was deleted.Therefore,Bna.C09ZEP and Bna.A09ZEP were likely candidate genes for OF1 and OF2,respectively.5.The transgenic complementation and overexpression experiments showed that the transfer of BnaA09.ZEP or BnaC09.ZEP alone can restore the orange flower color of O271 to yellow,and the carotenoid components in the petals of transgenic positive plants were similar to those of Y409.We also transformed two CRISPR/Cas9 constructs targeting both BnaA09.ZEP and BnaC09.ZEP into the yellow-flowered B.napus variety Westar.It was found that these two genes mutated in ten positive plants,and the flower color was changed to orange.The above results indicated that BnaA 09.ZEP and BnaC09.ZEP are the key genes controlling orange flower trait,and BnaA09.ZEP and BnaC09.ZEP have similar functions,indicating that BnaA09.ZEP and BnaC09.ZEP have a redundant function in carotenoid synthesis.6.The gene expression patterns of BnaC09.ZEP,BnaA09.ZEP and their two homologous genes BnaC07.ZEP and BnaA07.ZEP were studied by RT-PCR,qPCR and GUS assay.The results showed that BnaC09.ZEP and BnaA09.ZEP were mainly expressed in petals,while BnaC0 7.ZEP and BnaA 0 7.ZEP were mainly expressed in leaves,indicating that there are tissue-specific functional differentiations of multi-copy BnaZEP genes.7.By blasting the B.napus ZEP protein sequences,44 homologous sequences were obtained,and an evolutionary tree was constructed.In line with the results from other plants,BnaZEPs contain four functionally important regions.Brassica ZEPs were assigned to subclades in accordance with their locations on the A or C genomes.ZEPs translated from a locus on Brassica chromosome A07/C07 were located in a subclade and most closely related to the ZEP proteins from A.thaliana,forming a cluster that is sister to the ZEP from Brassica chromosome A09/C09.The results suggested that the divergence of Brassica ZEPs occurred earlier than the allopolyploidization event,and it is likely that Brassica ZEPs evolved rapidly after the whole-genome duplication and triplication event.8.To further study the effect of genes on flower color,we generated transcriptome profiles for buds and petals from O271 and two transgenic T2 homozygous strains,APSP and CPSP.As expected,most structural genes in carotenoid biosynthesis were transcriptionally regulated during flower development due to the introduction of BnaA09.ZEP or BnaC09.ZEP.The genes that control the synthesis of β-carotenoids were up-regulated,and that control the synthesis of α-carotenoids were down-regulated in flower tissues of APSP and CPSP.As a result,the violaxanthin of petals from APSP or CPSP increased significantly and the petals turned yellow.Furthermore,the introduction of BnaA09.ZEP or BnaC09.ZEP led to obviously reduced abundance of BnaA07.ZEP and BnaC07.ZEP in APSP and CPSP at all stages,indicating that BnaA07.ZEP and BnaC07.ZEP might up-regulated and partly compensate the function of BnaA09.ZEP and BnaC09.ZEP when the two genes were absent in O271.The results of KEGG and GO enrichment analysis showed that phenylpropanoid and flavonoid biosynthesis were the most enriched pathways.Most structural genes involved in flavonoid pathway were significantly upregulated at Petal1 and Peta12 stages.However,the altered flavonoid accumulation in petals is likely not the cause of color variation in this study.The altered flavonoid accumulation in petals may only influence the UV defensive ability and attractiveness to insect pollinators in B.napus flowers.