Functional Analysis Of BoaUGT74B1 And BoaAOP2 Genes In Glucosinolate Biosynthesis Of Chinese Kale

Glucosinolates are a diverse class of secondary metabolites that play a vital role in plant growth and development,defense response,and anticancer activity.The thiohydroximate S-glucosyltransferase（UGT74B1）is a crucial enzyme in production of the core gluosinolate structure,which can glycosylate thiohydroxamic acid to produce the corresponding desulfoglucosinolate.The 2-oxoglutarate-dependent dioxygenase（AOP2）plays an important role in the secondary side-chain modification of aliphatic glucosinolates,which engaged in the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates,such as the conversion of gluconapin（GNA）to glucoraphanin（GRA）.Chinese kale（Brassica oleracea var.alboglabra）is rich in glucosinolates,but contains low GRA content.This study focused on the functional analysis of Boa UGT74B1 and Boa AOP2.Boa UGT74B1 was transiently overexpressed by transient gene overexpression technology,and Boa AOP2 was edited by CRISPR/Cas9 technology and Chinese kale with high GRA content was obtained.The main findings are as follows:1.The coding sequence of Boa UGT74B1 single copy and Boa AOP2 three copies from Chinese kale cultivar‘Sijicutiao’were cloned.Bioinformatics analysis showed that Boa UGT74B1 belonged to glycosyltransferase superfamily and had highest homology with Bol UGT74B1 of broccoli,and Boa AOP2 belonged to the 2-oxoglutarate（2OG）and Fe（II）-dependent oxygenase superfamily and had the highest homology with Br AOP2 of Chinese cabbage.The subcellular localization showed that Boa UGT74B1,Boa AOP2.1,Boa AOP2.2,and Boa AOP2.3 proteins were located in chloroplasts,nucleus,chloroplasts,and chloroplasts of Chinese kale protoplasts,respectively.2.The results of spatial and temporal expression of Chinese kale cultivar‘Sijicutiao’showed that among different organs,Boa UGT74B1 has the highest expression level in young seeds;and among different flower tissues,Boa UGT74B1 has the highest expression level in pistils.The expression levels of Boa AOP2 three copies were different.In general,the expression level of Boa AOP2.1 gene was higher than that of Boa AOP2.3,and much higher than that of Boa AOP2.2.In different stages and organs,Boa AOP2.1 and Boa AOP2.3 had the highest gene expression in mature leaves.Among different flower tissues,the expression of Boa AOP2.1 was the highest in the pistils of blooming flowers,and the expression of Boa AOP2.3 was the highest in the sepals of closed flower buds.The expression of Boa AOP2.2 was low in the whole plant,and its expression was only detected in the cotyledon,young seeds,sepals of closed flower buds,and pistils of blooming flowers.3.The Agrobacterium-mediated transient gene overexpression system in Chinese kale was constructed,and Boa UGT74B1 was transiently overexpressed.In the transiently overexpressed plants white-flower cultivar‘Sijicutiao’and yellow-flower cultivar‘Fuzhouhuanghua’,Boa UGT74B1 has 1.53 to 2.93 times expression level higher than‘Sijicutiao’wild type,and 3.07 to 5.30 times expression level higher than‘Sijicutiao’wild type,respectively.The total indole glucosinolate content in Boa UGT74B1 transiently overexpressed plants was 1.30 to 7.11 times higher than‘Sijicutiao’wild type,and 2.13 to4.44 higher than‘Sijicutiao’wild type,respectively.In Boa UGT74B1 transiently overexpressed plants,each indole glucosinolate content was increased.In Chinese kale‘Sijicutiao’Boa UGT74B1 transiently overexpressed plants,4-methoxy glucobrassicin content was increased most,up to 12.14 times.In Chinese kale‘Fuzhouhuanghua’Boa UGT74B1 transiently overexpressed plants,glucobrassicin was increased most,up to22.76 times.4.Three Boa AOP2 copies were edited using CRISPR/Cas9,and 13 hygromycin-resistant plants of Target 1 and 14 hygromycin-resistant plants of Target 3 were obtained,and the mutation rate of Target 1 and Target 3 was 76.92%and 57.14%,respectively.Among the 10 mutants of Target 1,there were five aop2.1aop2.2aop2.3 triple mutants,two aop2.1aop2.2 double mutants,two aop2.1aop2.3 double mutants,and one aop2.1 single mutant.Three aop2.1aop2.2 double mutants and five aop2.1 single mutants were obtained by Target 3.Combined mutations were observed in Boa AOP2.1 and Boa AOP2.2,including large fragment deletions,insertions,and replacements,and only single base replacements were observed in Boa AOP2.3.Genetic analysis of the T1 generation showed that CRISPR-edited mutations were passed on to the next generation and partially re-edited.The content of GRA was 11.71 to 41.29 times(0.082 to 0.289μmol g^-1 FW)higher in boaaop2 mutants of the T1 generations than in the wild-type,and the ratio of GRA/GNA was increased,while the content of total aliphatic glucosinolates and GNA was decreased.