The Effects Of Acetylation Modification On Fatty Acid Metabolism In Brassica Napus L

Rapeseed is the largest self-produced oil crop in China.With the continuous improvement of national living standards,more and more quality of edible oil have been put forward,and the quality improvement has become a hot spot in the field of rapeseed breeding.Because of the traditional breeding techniques would take a long time,the molecular biological methods can be used to study the mechanism of fatty acid synthesis to speed up the breeding process.In previous research,Isobaric tags for relative and absolute quantification（iTRAQ）were used to analyze seeds of near-isogenic lines of high oleic acid B.napus from 20 to 35 days after pollination,and the four key differential proteins which related to fatty acid synthesis were all acetylated proteins.In this paper,the same materials were used for acetylation modification sequencing analysis,and western blotting（WB）and quantitative real-time PCR（qPCR）were used to verify the sequencing analysis results.A fatty acid metabolism-related protein corresponding gene BnaACP3 was screened and cloned,and a base mutation was introduced into its CDS sequence by overlapping primers PCR,and one of the amino acids was changed in a directed manner.Overexpression vectors of three genes(BnaACP3^63K、BnaACP3^63R and BnaACP3^63Q)were constructed and transformed into Arabidopsis thaliana.The specific results just as follows:1.The seeds of a near-isogenic lines with different high oleic acid B.napus（HOCR,oleic acid content 81.4%;LOCR,oleic acid content）from 20 to 35 days after pollination had been analyzed by acetylation sequencing.The B.napus line with low oleic acid content（LOCR）was used as for control,1.3 times was the change threshold and t-test p value<0.05 was the standard.Among the 2,473 sites of 1,409 proteins with quantitative information,80 sites were significantly up-regulated,and 21 sites were significantly down-regulated.After bioinformatics analysis,11 key differential proteins were screened:Three were related to fatty acid metabolism（GSBRNA2T00153661001,GSBRNA2T00054708001,GSBRNA2T00100854001）,four were related to glycolysis（GSBRNA2T00076479001,GSBR2T00069603001,GSBR2T008116001,GSBR2T00009340001）,three were related to photosynthesis（GSBR2T00134966001,GSBR2T000246）56001,GSBRNA2T00141918001)and one related to plant stress physiology（GSBRNA2T00129427001）.2.A differentially expressed histone（H3K27ac）was selected and verified by WB method with the same materials to verify the sequencing results.The expression in HOCR materials was higher than that in LOCR,which was consistent with the sequencing results;8 genes corresponding to differentially expressed proteins（BnaC03g33080D,BnaA06g36060D,BnaC09g16320D,BnaC09g07990D,BnaA01g30320D,BnaA02g27140D,BnaC04g33690D,BnaC09g29010D）were selected for qPCR verification with the same materials,and the expression levels of these 8 genes in HOCR materials were all higher than LOCR,which was consistent with the sequencing results.3.The corresponding genes of 11 different proteins（BnaC03g33080D,BnaA06g36060D,BnaC09g16320D,BnaA01g30320D,BnaA02g27140D,BnaC04g33690D,BnaC09g29010D,BnaC09g07990D,BnaA06g01050D,BnaA03g15830D,BnaA09g33250D）were selected to study the expression of leaves in bud and stalk stage,flowers in full bloom stage,and seeds in silique stage（20～35 d after pollination）by qPCR.The results showed that the expression levels of these 11 genes were higher than that of LOCR at seedling stage,5-6 leaf stage and mature stage,and increased first and then decreased from seedling stage to bud and moss stage,and the expression level at the 5～6 leaf stage was much higher than LOCR,which can be used for early screening;The expression levels of BnaA06g36060D,BnaC09g07990D,BnaA01g30320D,BnaA02g27140D,BnaC04g33690D,BnaC09g29010D,BnaA03g15830D,BnaA09g33250D genes in HOCR materials at 5 stages were higher than LOCR,indicating that the metabolic activities in HOCR might be higher than that in LOCR,which was expected to provide a reference for the study of fatty acid metabolism mechanism in B.napus.4.The BnaACP3^63K was obtained by PCR cloning with HOCR-seedling leaves,and a base mutation was introduced into the No.63 position of it by overlapping primer PCR method,after passing the sequencing test the BnaACP3^63R and BnaACP3^63Q with amino acid directed mutation were successfully obtained.Bioinformatics analysis showed that the homology of these 3 base sequences with the published sequence of the database was more than 98%,the amino acid sequence was more than 99%,the encoded amino acids were all 131,the molecular weight was about 14.1 k Da,the instability coefficient was greater than 40.The electrical points were all below 5,which were unstable hydrophobic acidic proteins and were located on the chloroplast without transmembrane structure.The prediction of level 3 model indicated that all the three proteins belonged to PP-binding super family.Clusters analysis of amino acid sequences showed that it was closely related to Arabidopsis thaliana,Lyceum luteolata,Capsella bursa-pastoris and flax mustard.5.The plant expression vectors pCAMBIA1300-BnaACP3^63K,pCAMBIA1300-BnaACP3^63Rand pCAMBIA1300-BnaACP3^63Q were constructed by double digestion.The results showed that the vectors were successfully constructed by PCR and double digestion.The three expression vectors were transformed into competent Agrobacterium tumefaciens and transformed into Arabidopsis thaliana.