Cloning And Functional Analysis Of BnAPX1 Gene In Brassica Napus

Rapeseed is the oil crop with the widest regional distribution and the largest sowing area in China,which plays an important role in China’s agricultural and social economic development.Under stress,rapeseed produces a large number of reactive oxygen species,resulting in oxidative stress,which affects the yield and quality of rapeseed.Ascorbate peroxidase（APX）is a key enzyme for scavenging reactive oxygen species in plants,using ascorbate（As A）as an electron donor to convert H₂O₂into H₂O,thus preventing plants from being poisoned by reactive oxygen species and playing an important role in plant resistance to oxidative stress.BnAPX1 is a member of APX gene family.In this study,BnAPX1 gene was cloned and its function was preliminarily studied.（1）CDS sequences of APX1 gene was cloned from Brassica napus Zhongshuang 11 by RT-PCR.Bioinformatics analysis showed that BnAPX1 contains the characteristics of the APX family:heme binding site,substrate binding site and K⁺binding site.It is a member of Plant＿peroxidase＿like Superfamily,and was highly homologous to other plants’APX.（2）Tissue specificity analysis showed that the expression pattern of BnAPX1 liked leaf>root>stem>flower>pods in Zhongshuang 11.（3）Biotic and abiotic stress expression analysis.The expression of BnAPX1 could be induced and promoted in drought,high temperature,low temperature and salt stress.After inoculation with Sclerotinia sclerotiorum,the expression of BnAPX1 was first inhibited,then increased and then decreased.（4）BnAPX1 overexpression vector p BI121-BnAPX1 was constructed,and was transformed into Arabidopsis thaliana and Brassica napus Zhongshuang 6.Screened T2generation of transgenic Arabidopsis thaliana seedlings,and obtained 2 strains T0generation of transgenic rapeseed seedlings.Fluorescence quantitative analysis showed that the expression of this gene in 2 strains T0 transgenic rapeseed plants was 3.9 times and 0.9times higher than CK,respectively.Sclerotinia sclerotiorum inoculation infection experiments showed that the lesion area of T0 transgenic rapeseed seedlings was no significant difference between T0 and CK.Resistance identification experiments showed that the overexpression of BnAPX1 could not improve the resistance to Sclerotinia sclerotiorum.（5）BGK01-BnAPX1 knockout vector was successfully constructed and transformed into Brassica napus Zhongshuang 11,and obtained 11 strains T0 generation gene editing plants.Genotypes of the 11 strains gene editing plants were detected,and two of them mutated at the target sites.