The Studies On GH3 Gene Family And The Gene Expression Regulation During Polyploidization In Brassica

Polyploidization has been regarded as an important evolution force to promote plant species diversity,and all flowering plants have undergone one or several whole genome duplication events during the process of evolution.The changes in plant genomes and gene expression were caused by polyploidization,which make polyploids more adaptive to the environment,helping them evolve in a stable direction.In this study,Brassica tetraploid and its ancestral species,and Brassica hexaploid and its parents were used as experimental materials to investigate the effects of alloploidization on the structure and expression level of GH3 gene,pre-mRNA alternative splicing pattern and lncRNA expression level and illustrate how lncRNAs regulate protein-coding gene expression in allopolyploid.The GH3 gene family is involved in numerous growth and development processes associated with phytohormone.A total of 38,25 and 66 GH3 genes were confirmed in Brassica rapa,Brassica oleracea,and Brassica napus,respectively.The GH3 genes were unevenly distributed on the chromosomes of B.napus with 39 on A_n and 27 on C_n,which was similar to the number of GH3 genes on the B.rapa and B.oleracea genomes.Allopolyploidization allowed the GH3 gene family to expand in B.napus,and six new GH3 genes are produced.Compared with ancestor species,the exon-intron compositions of the most GH3 genes remained unchanged,but the cis-element types of the promoter regions obviously changed,and the expression patterns also significantly changed in B.napus.Alternative splicing is an important mechanism for regulating gene expression levels and protein diversity,and plays a vital role in many biological processes.To research how allopolyploidization affects the AS patterns of pre-mRNAs,we carried on a genome-wide AS analysis of pre-mRNAs in Brassica hexaploid and its parents.A total of 7913,14447,and 13205AS genes were found in Brassica rapa,Brassica carinata,and Brassica hexaploid,respectively.Fifty-six differently spliced genes were identified between Brassica hexaploid and its parents.Alternative 5'splice sites were frequently located 4 bp upstream of the dominant 5'splice sites,and the alternative 3'splice sites were usually located 3 bp downstream of the dominant 3'splice sites in Brassica hexapliod,which was similar to maternal parent.Some pre-mRNAs in Brassica hexaploid had the alternative splicing types different from parents,and other pre-mRNAs had the same alternative splicing types as parents,but the splicing sites were different from parents.In addition,920 new AS genes were discovered in Brassica hexaploid,and these genes were significantly enriched in‘cellular aromatic compound metabolic process',‘cellular nitrogen compound biosynthetic process'and‘aromatic compound biosynthetic process'.The pre-mRNA alternative splicing analysis of the non-additive genes in Brassica hexaploid showed that the pre-mRNAs of the down-regulated non-additive genes were more likely to undergo alternative splicing,suggesting that alternative splicing may be involved in the regulation of non-additive gene expression.By analyzing the results of TA cloning and Sanger sequencing,we found that the pre-mRNAs of GRP7/8 genes had an alternative splicing type different from previous report,namly new intron,and the appearance of this alternative splicing type may indicate the formation of a novel mechanism for regulating GRP7/8 gene expression.lncRNAs affect gene expression through a variety of regulatory mechanisms and participate in many biological processes related to plant growth and development.We identified 2725,1672 and 2810 lncRNAs in B.rapa,B.carinata,and Brassica hexaploid,respectively.There were 725 differentially expressed lncRNAs between Brassica hexaploid and its parents,and the expression patterns of these differentially expressed lncRNAs were divided into six categories,showing the diversity of lncRNA expression in Brassica hexaploid.The lncRNAs presented paternal parent-biased and non-additive expression pattern in Brassica hexaploid.lncRNAs were discovered to directly regulate protein-coding gene expression by cis or trans-action.Most target genes were positively regulated,and they were mainly enriched in the metabolic process,exposure to stimulus,reproduction,biological regulation and so on,showing that lncRNAs may play important roles in these biological processes.Finally,lncRNAs could also indirectly regulate protein-coding gene expression as precursors or targets of miRNAs.We found that 61 lncRNAs served as potential targets for 100miRNAs,and 15 lncRNAs served as precursors for 15 miRNAs,suggesting that lncRNAs may be involved in miRNA-mRNA interaction networks.At the same time,we also found some conserved motifs of lncRNAs,and the conserved motifs may be the key to the lncRNA regulatory function.These findings provided a reference for further studying the function and action mechanisms of lncRNAs in polyploid plants.In summary,the occurrence of these changes in Brassica allopolyploids make them adapt to a wider range of environmental conditions.These results laid the foundation for further exploration of the molecular mechanisms of polyploid plant evolution and adaptability.