Preliminary Functional Study On Cytochrome B₅ Gene BnaA02g10220D In Brassica Napus L.

Cytochrome b₅is an amphiphilic heme-binding protein,which is an important redox protein in organism.Plant cytochrome b₅has various physiological functions.It can be used as an electronic carrier to participate in a series of important REDOX reactions in plants,such as fatty acid desaturation.It can also be used as a protein allosteric activation regulatory protein to participate in sugar signaling and ethylene signaling.However,there are few studies on cytochrome b₅,in rape.Our group has previously screened out a down-regulated expression gene BnaA02g10220D within the genetic mapping interval of a young leaf etiolation mutant in Brassica napus.The BnaA02g10220D is homologous to AT5G53560,the gene encoding cytochrome b₅isoform E in Arabidopsis thaliana.No studies on BnaA02g10220D have been reported in rape.In order to identify the relationship between BnaA02g10220D and the phenotype of young leaf etiolation and preliminarily understand the function of BnaA02g10220D gene in Brassica napus,bioinformatics analysis,subcellular localization and gene expression characteristics of BnaA02g10220D were carried out in this study.BnaA02g10220D overexpression transgenic plants of ZS11 and young leaf etiolation mutant K189 in Brassica napus were obtained.The overexpression transgenic plants were observed in leaf color and were analyzed in high temperature tolerance and contents of oleic acid,linoleic acid and linolenic acid in seeds of T₁generation and leaves of T₂generation.The main research results are as follows:1.A total of 20 genes encoding cytochrome b₅were screened from the rape database,and they were divided into two categories according to their genetic relationships:Class I and Class II.BnaA02g10220D belongs to Class II and is closely related to At CB5-E,one of the five cytochrome b₅isoforms in Arabidopsis thaliana.Both BnaA02g10220D and At CB5-E are single transmembrane unstable hydrophilic proteins.2.The c DNA and CDS sequences of BnaA02g10220D gene could be cloned from Brassica napus ZS11 of,but not from the young leaf etiolation mutant.3.The subcellular localization vector of BnaA02g10220D gene was constructed,and the subcellular localization was conducted by protoplast transformation of Arabidopsis thaliana.It was found that BnaA02g10220D protein was located in the endoplasmic reticulum,which was consistent with the prediction result of bioinformatics.4.BnaA02g10220D was expressed in all kinds of organs in Brassica napus,but the expression level was different.The expression level of BnaA02g10220D was the highest in siliques at 50 days after flowering,followed by that in root and seeds at 50 days after flowering,and the lowest in epicotyl and hypocotyl.The results of hormone and stress treatment showed that the expression of BnaA02g10220D was significantly up-regulated after SA,low temperature and high temperature treatment,and significantly down-regulated after ABA,Me JA and ACC treatment.These results indicated that BnaA02g10220D had differential functions in various tissues and was involved in hormone signal transduction and stress response in Brassica napus.5.The overexpression vector of BnaA02g10220D gene was constructed,and the overexpression transgenic plants of ZS11,a cultivar with green leaves,and the young leaf etiolation mutant K189 in Brassica napus were obtained,which provided materials for analyzing the function of BnaA02g10220D gene in Brassica napus.6.The fluorescence quantitative PCR showed that the expression of BnaA02g10220D in the transgenic plant of young leaf etiolation mutant K189 was 10-20 times higher than that of untransformed K189,but the phenotype of young leaf etiolation did not recover.This indicates that BnaA02g10220D may not be the objective gene for young leaf etiolation.7.Under high temperature stress,the PRO accumulation level in BnaA02g10220D overexpressed plants was higher than that of wild type,and the MDA accumulation level was lower than that of wild type,indicating that overexpression of BnaA02g10220D gene could reduce the degree of lipid peroxidation of cell membrane,increase the accumulation of proline,and improve the stress resistance and reduce the damage.8.In seeds of T₁generation of BnaA02g10220D overexpressed plants the oleic acid content was increase in significance than that of wild type,the linoleic acid content was significant reduction than that of wild type,and the linolenic acid content was increase or decrease than that of wild type.There was no significant difference in the contents of oleic acid,linoleic acid and linolenic acid in the T₂generation leaves of BnaA02g10220D overexpressed plants compared with the wild type.These results indicated that overexpression of BnaA02g10220D could increase oleic acid content in seeds,which provided a new idea for high oleic acid breeding in rapeseed.The present study can provide valuable reference for the functional research and application of Cytochrome b₅in rape.