Brassica Napus Acyl-CoA-bing Protein 1(BnACBP1) Gene Function Research

Canola is a dominant oilseed crop in China.Notwithstanding,canola production still face serious limitations.for example,in southern China it forms a crop rotation planting pattern with rice,but the extended growth period of Brassica napus at present affect rice cultivation and can expose canola to biotic and abiotic stresses,thus limiting the efficient use of land.Therefore,it is essential to develop new and stable Brassica napus varieties with shorter growth period,high yielding,superior oil content,disease resistance and suitable for mechanized harvesting.In this study,we set out to identify useful genes for canola,by developing an inducible gain functional mutant library of Arabidopsis thaliana by iFox-Hunting method using canola seeds cDNA and a mutant rsl-1327 with leaf senescence phenotype was identified from a mutant screen.The transgene insertion in the mutant was studied,including the transcriptional response resulting from transgene induction.Finally,the possible mechanism of early senescence phenotype of the mutant was discussed.This study provides theoretical guidance for gene discovery and functional characterization of useful genes in canola.The main results are as follows: 1.First the leaf senescence phenotypes of the mutant rsl-1327 was characterized.We found that the leaf senescence phenotype appeared about 4 weeks after the inducer treatment.Its aging rate,flowering period,and mature period were significantly different from wild type Arabidopsis control.At the same time,there was a significant difference in both the physiological level of chlorophyll content and the molecular level of senescence marker gene expression.2.To further characterize the mutant,we cloned the inserted gene of the mutant rsl-1327 and analyzed the 3D structure of the amino acid sequence.The result showed that the protein has a transmembrane domain which is mainly composed of α-helix and a secondary protein structure.DNA sequence alignment showed that the inserted gene is highly homologous to ACBP1 of ACBP family.Because the mutant gene was derived from canola,we compared the protein sequence of the gene with canola and Arabidopsis ACBP1 protein sequences and analyzed the functional domains of the protein in depth and found that their protein has a functional domain which are highly conserved.Further analysis revealed that the transgene had high similarity with BnaA02g10270D(BnACBP1-2)on the A2 chromosome of Brassica napus genome,therefore we speculate that the minor differences could be responsible for their phenotypic difference.3.To further determine whether the mutant leaf senescence phenotype resulted from the inserted gene,we cloned the transgene and re-introduced it into Arabidopsis thaliana using a 35 S promoter construct.The result showed that 35 s BnACBP1-2 transgenic lines had the early senescence phenotype in leaves,and the degree of senescence was comparable to the expression of 35sBnACBP1-2.Subsequently,we selected two transgenic lines to determine their chlorophyll content and the expression of the senescence marker gene.The result showed very low chlorophyll content and high level of senescence.At the protein level,we used GFP antibody by Western Blot to confirm that the BnACBP1-2 protein was indeed expressed in the transgenic lines with the leaf premature senescence phenotype.4.We then analyzed the subcellular localization of the transgene through protoplast transformation.Similarly,the expression pattern and fatty acid composition of two ACBP1 genes were evaluated.The result of the subcellular localization showed that the rapeseed ACBP1 gene was localized in the endoplasmic reticulum and plasma membrane of protoplasts,similar to Arabidopsis ACBP1.The expression of BnACBP1 in different tissues and stages of canola showed that the expression level of BnACBP1 was high in the later stage of seed development,roots and buds.Therefore,the promoter regions of two ACBP1 genes were separately cloned and expressed using GUS constructs.The results revealed GUS activity in mature embryos,seedling roots,and mature pollen was consistent with the distribution of BnACBP1 expression.At the same time,the GUS staining analysis showed that wounding in leaves could trigger GUS activity after treatment.Therefore,we examined GUS activity before and after injury treatment.The result showed normal GUS expression in healthy leaves however it was significantly increased after injury treatment.Since the BnACBP1 protein is involved in fatty acid transport,we also determined the composition of fatty acids in the BnACBP1-2 overexpression transgenic line and found that the content of 18-carbon unsaturated fatty acids in the leaves increased.These differences with Arabidopsis ACBP1 suggest that the ACBP1 gene of canola may function differently to regulate early senescence phenotype.5.So far,we have shown that the overexpression of BnACBP1-2 may lead to early senescence in leaves.Next,we combined our data with transcriptome sequencing analysis to find genes that may responded to inducible expression of BnACBP1-2.The transcriptomic data,showed that there were significant differences in the expression of genes related to linolenic acid metabolic pathway and jasmonic acid metabolic pathway.In order to verify this result,we determined the content of jasmonate and oxidized lipids in the 35sBnACBP1-2 transgenic line and found that the content was significantly increased and the expression of BnACBP1 was induced by jasmonic acid treatment.In contrast,the phenotype of early senescence of leaves was inhibited to some extent in the background of the mutant lox3,lox4,coi1,suggesting that BnACBP1-2 mediates early senescence phenotype through apparent induction of jasmonic metabolic pathway.Since jasmonic acid plays an important role in disease resistance,we challenged 35sBnACBP1-2 transgenic line with Botrytis cinerea and found that the transgenic strain has a certain resistance to Botrytis cinerea.In summary,we speculate that the over-expression of BnACBP1-2 led to early senescence in leaves probably due to the possibility that it increased the transport of unsaturated fatty acid from the ER to the plastids thereby increasing the content of linolenic acid in the plastids.The likely scenario would be that increased linolenic acid will be oxidized to oxidized fatty acids and further synthesized into jasmonic acid which led to the accumulation of jasmonic acid and triggered the downstream signal response to cause senescence.Interestingly,BnACBP1 expression can be triggered by injury and jasmonic acid treatment,and it is well known that jasmonic acid is modulate plant pest and disease resistance.Therefore,the study of ACBP1 in rapeseed is very useful to understand the effect of fatty acid transport on plant growth and development;biotic and abiotic stress resistance.This study is of great theoretical significance to canola breeding and production.