| Non-heading Chinese cabbage(Brassica campestris ssp.chinensis Makino),originated in China,is an important Brassicaceae vegetable.Self-incompatibility(SI)in phanerogam is a genetic mechanism that promotes outcrossing and prevents inbreeding,which is formed during the long evolutionary period.The utilization of SI is a major approach for hybridization seed production in Brassicaceae vegetable crops.SI is also an excellent model system for cell-cell communication and signal transduction in higher plants.However,the mechanism of SI is largely unknown.Here,we construct SCP stigma-specific expression vector,and studied the effect of this gene on pollen germination;we characterized the transcriptome of stigma at 10 minutes after compatible or incompatible pollination;candidate reference genes which is relate to self-incompatibility were selected in non-heading Chinese cabbage;The BrPUB gene family was analyzed by using the genomic information of Chinese cabbage.The main findings are as follows.1.Functional analysis of Brassica campestris SCP geneWe used the stigma-specific SLR1 promoter to construct BcSCP expression vector,digestion results showed that SCP vector was successfully constructed,the construct was then mobilized into Agrobacterium GV3101,followed by Agrobacterium-mediated transformation of wild-type Arabidopsis plants using the floral dip method,PCR identification showed that the carrier has been successfully integrated into the Arabidopsis genome.Laser scanning confocal microscopy was used to observe the expression of green fluorescent protein in the stigma.The fluorescence microscopy analysis of T1 transgenic Arabidopsis showed that overexpression of BcSCP had no effect on the germination and elongation of pollen tubes.2.Transcriptome analysis of stigma at early stage after compatibility or incompatibility-pollination in non-heading Chinese cabbageIn order to identify key genes of pollen-stigma interaction in non-heading Chinese cabbage,we characterized the transcriptome of stigma at 10 minutes after compatible or incompatible pollination by using compatible and self-incompatible lines in non-heading Chinese cabbage.On average,65 million thousand original sequences were obtained for each sample,with an average of 72.5%mapped to the Chinese cabbage genome.The differentially expressed genes(DEGs)were analysized and finally a total of 511 DEGs were identified.Among them,the expression of the 493 genes was up-regulated and the expression of the 18 genes was down-regulated.Functional annotation and metabolic pathway analysis of DEGs indicated that compatibility reaction and incompatibility reaction involved different pathways.We analyzed the transcriptome of stigma at early stage after compatibility or incompatibility pollination in non-heading Chinese cabbage,this will provide rich resources for our further research on compatibility and self-incompatibility.3.Identification and validation of reference genes in non-heading Chinese cabbage flowersIn this study,13 candidate reference genes were selected and examined systematically in compatible and self-incompatible lines of non-heading Chinese cabbage.Two RT-qPCR analysis software,geNorm and NormFinder,were used to evaluate the expression stability of these genes systematically.Results revealed that best-ranked references genes should be selected according to specific sample subsets.DNAJ,UKN1 and PP2A were identified as the most stable reference genes among all samples.Moreover,our research further revealed that the widely used reference genes,CYP and ACP,were the least suitable reference genes in most non-heading Chinese cabbage flower sample sets.To further validate the suitability of the reference genes identified in this study,the expression level of SRK and Exo70Al genes which play important roles in regulating interaction between pollen and stigma were studied.Our study presented systematic study of reference gene(s)selection for SI study and laid a foundation for further study the mechanism of SI in non-heading Chinese cabbage.4.Genome-wide analysis and identification of PUB gene family in Chinese cabbagePUB genes are important target genes which are thought to play important roles in self-incompatibility.We systematically and comprehensively analyzed the PUB gene family of Chinese cabbage,101 BrPUB genes were identified from Chinese cabbage and were anchored onto the 10 chromosomes and three subgenomes.By specific protein domains and a phylogenetic analysis,the B.rapa PUB(BrPUB)gene family was subdivided into 10 groups.We selected 15 representative plants and analyzed the evolution of PUB gene with the addition of Chinese cabbage.Chinese cabbage and Arabidopsis shared 22 orthologous gene pairs,and the expansion of BrPUB genes mainly resulted from genome triplication.Moreover,we analyzed the expression patterns of BrPUB genes in four tissues based on RNA-seq data.Altogether,we analyzed the PUB gene family of Chinese cabbage,this will provide rich resources in our understanding of PUB genes in self-incompatibility. |
