| Brassica napus.L,belonging to Brassica of Cruciferae,is the one of major oil crops in the world.The branches of rape is the important agronomic trait,which is closely related to yield grain.As we know,the branches are derived from axillary buds.However,the axillary buds of rape,which grown in the field,can not develop into branches totally,especially the lower and middle axillary buds.This phenomenon significantly results in the loss of rapeseed yield grain.In order to explore the reason why the lower and middle axillary buds can’t develop into branches,a transcriptome analysis containing the axillary buds at the top,upper,middle and lower parts of B.napus budding stage was done in this study,and the key factors regulating the growth of axillary buds were excavated and performed functional verification.The main results are as follows:1.Transcriptome sequencing of axillary buds in different parts.Transcriptome sequencing was performed on axillary buds in different parts of rape at the budding stage.Using the top part as a control,27992 differentially expressed genes(Degentially Expressed Genes,DEGs)were obtained based on | log2(foldchange)| ≥1,FDR <0.001.The number of DEGs at the top vs.the bottom is the largest with21,906.And,the DGEs were enriched from three aspects by GO analysis,including BP(biological processes),MF(molecular functions),and CC(cellular components).The GO results showed that DEGs were mainly enriched into catalytic activity and cellular components,belonging to cellular processes.The results of KEGG enrichment analysis showed that DEGs are mainly enriched in carbon metabolism,amino acid biosynthesis,plant hormone signal transduction,photosynthesis and starch sucrose metabolism.What’s more,we selected 464 DEGs related to axillary bud growth and development,including 101 DEGs related to sugar signaling,80 DEGs encoding the genes related to hormone signaling,166 DEGs encoding transcription factors and 117 DEGs related to photosynthesis.As for the sugar signaling pathway,most of BnSPP and BnSPS,which participates in encoding the key enzymes for sucrose synthesis,are down-regulated in the middle and lower part of axillary buds.Among the genes related to the sugar signaling pathway in the middle and lower parts,the expression of genes that inhibit the growth of axillary buds is up-regulated and the expression of genes that promote the growth of axillary buds were down-regulated.Most of the hormonal-related genes identified were related to abscisic acid signaling,and most of them were up-regulated in the middle and lower parts.As for the transcription factors related to axillary bud growth and development,a total of 6 transcription factors were identified,containing BnPIF,BnHB-1,BnLSH4,BnBRC1,BnWRKY,and BnWD40,respectively.In Arabidopsis,At BRC1 can inhibit the growth of axillary buds.In this study,compared with the top part,the expression of BnBRC1 was up-regulated in the middle and lower parts,and the difference was up to 11 times,which may be an important reason that axillary buds cannot develop branches in the middle and lower parts.In addition,the expression levels of 15 candidate genes in the qRT-PCR verification experiments were consistent with the results of transcriptome sequencing.2.Determination of soluble sugar content of axillary buds in different parts of B.napusThe anthrone method was used to determine the soluble sugar content of the axillary buds of the top,upper,middle and lower parts of rape.The results showed that the soluble sugar content decreased from the top to lower,and the soluble sugar content of the top axillary buds was nearly 3 times than it in the lower.Besides,there are extremely significant differences between the different parts.3.Bioinformatics analysis of BnTCP members.In B.napus,there are 75 members of the BnTCP gene.Among the 75 members,the longest CDS sequence of BnTCP13 d is 1290 bp and encodes 429 amino acids,while the shortest CDS sequence of BnTCP21 a is 531 bp and encodes 176 amino acids.Promoter analysis of the BnTCP family showed that 14 BnTCP members contained circadian rhythm regulatory elements,and most of them contained cis-acting elements that respond to plant growth and development and respond to plant hormones.According to the transcriptome results of a cultivar named “ZS11” in rape,the BnBRC1 gene family was specifically expressed in axillary buds,and the expression level was lower in roots,leaves,stems and cotyledons(without embryos).4.BnBRC1 gene function verification Based on the CDS sequence of BnBRC1,the specific primers were designed and amplified with a 855 bp fragment.And the overexpression vector p Cambia2301M1 B was constructed by a double-enzyme digestion method and then transformed genetically to to prepare for genetic transformation of Arabidopsis thaliana.The positive trans-gene plant was filtered by antibiotic screening and identified by molecular method.In order to observe which part of BnBRC1 is located in the cell,we first performed a subcellular localization prediction.The results indicate that the gene may be located in the nucleus.In order to further detect its expression site,the CDS sequence of BnBRC1 was cloned and the p EGAD vector was constructed with CDS sequence by a double-enzyme digestion method and planned to transferred into tobacco to observe the expression site. |
