| Orphan genes have no significant sequence similarity to previously identified genes,which are widely found in various organisms.Orphan genes have been confirmed to be involved in substance metabolism,response to biotic and abiotic stresses,influence species-specific evolution and species-specific traits,and show high specificity in some tissues,organs or developmental stages.With the continuous upgrading of sequencing technology,the genetic information of many species has been revealed,and scholars attach great importance to the study of orphan genes.The release and update of genomic information of B.rapa and other plants in genus Brassica provides an important prerequisite for the identification of B.rapa orphan genes.In this study,orphan genes were screened and identified for the first time by using the reference genome of B.rapa,and the classification,gene features,specificity and expression patterns of orphan genes were systematically analyzed;the Brassica campestris orphan genes over-expression transgenic library was created in Arabidopsis thaliana,and the phenotype of transgenic plants were comprehensively investigated;the orphan genes transgenic plants were screened with stresses responsive and soluble sugar content variations;and one of the transgenic plants was used as an example to explore the mechanism of the effect of orphan genes on the variation of soluble sugar.This study laid a good foundation for further study on the function of Brassica campestris orphan genes and provided excellent materials and gene sources for genetic improvement of Brassica campestris crops.The primary findings are as follows:1.Screening and classification of orphan genesIn this study,the orphan genes in B.rapa genome(v2.5)were systematically screened for the first time.We identified 1540 Brassica-specific genes(BSGs),1824 Cruciferae-specific genes(CSGs)and 45462 evolutionary-conserved genes(ECGs).Compared with ECGs,BSGs and CSGs have fewer multiple exon genes,higher GC content and shorter gene length.By further classifying BSGs,529 Real A subgenome-specific,474 A subgenome-specific,130 A/B subgenome-specific,229 A/C subgenome-specific and 178 A/B/C subgenome-specific type BSGs were obtained.2.Validation of orphan genes145 out of 529 Real A subgenome-specific BSGs were identified by PCR using 51 inbred materials from Brassica and Cruciferae.The results showed that 145 BSGs did not exist in wild type A.thaliana,and 52 BSGs were successfully amplified in B.rapa reference genome sequencing material 'Chiifu'.49 materials from six Brassica genomes were used to verify the 52 BSGs,results indicated that A/B/C subgenome-specific type accounted for the most,Real A subgenome-specific type accounted for the least.3.Analysis of orphan genes expression patternsExpression pattern validation of 1540 BSGs using reassembled transcriptome data,the results showed that 73 BSGs were expressed in different tissues of B.rapa,and 180 BSGs were expressed in Chinese cabbage treated by Plasmodiophora brassicae.The results of semi-quantitative PCR analysis of 52 Real A subgenome-specific BSGs successfully verified in 'Chiifu' showed that 20 genes were expressed in different developmental stages or tissues of Chinese cabbage,41 genes were expressed in susceptible Chinese cabbage materials infected by P.brassicae for ten days,and 39 genes were up-regulated.BSGs showed tissue,organ and stage-specific expression,and some genes could be induced by P.brassicae.4.Construction of Brassica campestris orphan genes transgenic library128 Brassica campestris orphan genes were successfully transformed into Arabidopsis by floral-dip method,and the Brassica campestris orphan genes over-expression transgenic library was constructed.These genes were named Brassica rapa orphan genes(Br OGs).Ten Arabidopsis transgenic plants were randomly selected for validation at the level of DNA and expression.The results showed that both gene and coding sequences in the transgenic plants could be successfully amplified.After sequencing,the correctness of the transgene was verified,and no target bands were found in the wild type.5.Phenotypic investigation of Brassica campestris orphan gene transgenic plantsThe phenotype of T2 homozygous mutants in the transgenic library were investigated,the results showed that 73% of Br OGs OE transgenic plants had phenotypic variations,including leaf shape,flowering time,stem height,rosette radius,leaf color,silique length and seed number.Nearly 50% of the transgenic plants showed delayed flowering,and these transgenic plants were accompanied by many other phenotypic variations.The morphological changes of the transgenic plant leaves included wavy leaves,serrated edges,hairy leaves,upward or downward curved leaves and increased number of rosette leaves,which indicated that there was a significant correlation between the leaf shape of these mutants and that of Chinese cabbage.6.Screening of stresses responsive transgenic plants24 randomly selected Br OGs OE transgenic plants with no significant phenotypic difference were treated by Pseudomonas syringae pv.tomato(Pst)strain DC3000 stress,salt stress and heat stress.The results showed that three transgenic plants were resistant to the infection of Pst DC3000,high transcription level of At PR1 and fewer bacteria were detected in the transgenic plants;12 mutants were not sensitive to salt stress,five transgenic plants were salt sensitive,seven transgenic plants were salt resistant;11 transgenic plants were not significantly different from the wild type in response to heat stress,and the survival rate of 13 transgenic plants significantly decreased under heat stress.7.Screening of Arabidopsis transgenic plants with variable soluble sugar43 Br OGs OE transgenic plants were randomly selected for soluble sugar content analysis.The results showed that the soluble sugar content of 42 transgenic plants changed in different degrees,among which 17,29,20 and 34 transgenic plants with significant increase in fructose,glucose,sucrose and total soluble sugar,4,1,10 and 1 transgenic plants with significant decrease in content,and 22,13,13 and 8 transgenic plants without significant change in content.8.Function analysis of Br OG1Using CRISPR/Cas9 technology to knockout Br OG1A(Bra A08002322)and its highly homologous Br OG1B(Bra Sca000221),the 'brog1' homozygous transgenic plant was obtained.There was no significant phenotypic variation between the T2 'brog1' transgenic plants and wild type 'GT-24',and there was no significant phenotypic difference between the 'Br OG1AOE' transgenic plants and the Arabidopsis wild type.The content of Fru,Glc and total soluble sugar in 'Br OG1AOE' transgenic plants were significantly higher than those in wild type,and Suc content significantly decreased;Compared with wild type,the content of Fru,Glc and total soluble sugar in 'brog1' transgenic plants were significantly decreased,and Suc content were significantly increased.Compared with WT,SUS activity and the expression of At SUSs were significantly decreased in 'Br OG1AOE' transgenic plants,and there was no significant difference in INV activity;there was no significant difference in INV activity between 'brog1' transgenic plants and wild type,but SUS activity was significantly increased,and the expression of Br SUS1 b,Br SUS3 and Br SUS5 were significantly increased.The results showed that the 'brog1' transgenic plants complemented the 'Br OG1AOE' phenotype,and the analysis showed that the Br OG1 may affect the soluble sugar metabolism in a SUS-dependent manner. |
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