De Nove Transcriptome Of Chinese Kale And Functional Identification Of BocODD1/2

Glucosinolates are a group of secondary metabolites,mainly found in the Brassica species,and involved in various plant responses,including stress resistance,insect resistance,disease resistance,etc.,and also participates in human anti-cancer and anti-oxidation.The content of glucosinolates in various tissues is different,but there is currently no systematic study on the glucosinolate metabolic genes in various tissues to date.ODD regulates 3-butenyl glucosinolate(NAP)to 2-hydroxy-3-butenyl glucoside(PRO)in Arabidopsis,the main source of bitterness in Brassica,and its degradation products can cause goiter in pig body,research on ODD can help to improve the nutritional value of plants.In this study,the expression of glucosinolate-regulated genes in 11 tissuse of Chinese kale(Brassica olearcea var.chinese Lei)were analyzed by RNA-Seq technology,we found ODD were significantly expressed in roots,and choose to further study.The researches are as follows:(1)By sequencing the transcriptomes of 11 different tissues of Chinese kale including senescent leaves(SL),mature leaves(ML),young leaves(YL),leaf veins(LV),petioles(Pe),young bolting stem flesh(YB),middle bolting stem flesh(MB),bolting stem skin(BS),flower buds(FB),combining sites(CS),and roots(Ro),a series of differentially expressed genes were obtained.Analyzed the expression of Unigenes-73909(MAM1/2),Unigenes-38232(IPMDH1),Unigenes-55852(BCAT-4),Unigenes-70138(CYP79F1),Unigenes-71114(CYP83A1),Unigenes-71455(CYP83B1),Unige nes-76549(SUR1),Unigenes-45871(STb),Unigenes-57901(FMOGS-OX1),Unigenes-71928(IGMT2),Unigenes-106064(GSL-OH)and Unigene-34010(GSL-OH)by q RT-PCR,the results were consistent with the RNA-Seq.(2)Two ODD homologous genes location on GSL-OH that are significantly expressed in roots were further study,according to the sequence obtained by RNA-Seq(Unigene＿34010 and Unigene＿106064),three homologous genes Boc ODD1,Boc ODD2 and Boc ODD3 were cloned from Chinese Kale.they belong to the 2-oxoglutarate/Fe(II)dependent dioxygenase gene family and has a typical 2-oxoglutarate/Fe(II)dependent dioxygenase conserved domain.The ORF of Boc ODD1 gene is 1077 bp in length,encoding 358 amino acids.the relative molecular mass of protein is 40230.23 k Da and the theoretical isoelectric point is 6.87.The length of ORF of Boc ODD2 is 1077 bp,encoding 358 amino acids.the relative molecular mass is 40182.08 k Da and the theoretical isoelectric point is 6.19.The ORF of the Boc ODD3 is 801 bp encoding 266 amino acids,the relative molecular mass is 30122.56 k Da and the theoretical isoelectric point is 5.27.The homology among the three gnes was over 90%,and Boc ODD1 and Boc ODD2 were over 95%.Three Boc ODDs have different homologies with the ODD in cruciferous family.Boc ODDs have closer genetic relationship with ODDs from Brassica rapa and Brassica oleracea than Brassica napus.(3)The content of glucosinolates in different tissues is varied.In order to identify the relationship between genes expression levels and glucosinolates content,Boc ODD1/2 were cloned from Chinese Kale Shengzhong(high PRO/NAP value)and Taoshanzhonghua(low PRO/NAP value),and the sequence,expression pattern of Boc ODD1 and Boc ODD2 and glucosinolates content were analyzed.There was no difference in the gene sequence of Boc ODD1 and Boc ODD2 between two varieties.Treated with Me JA,IAA and GA3,the expression level of Boc ODD1 and Boc ODD2 were up-regulated,the ration of PRO/NAP value were increased,which may result in the difference in glucosinolates content.The expression patterns of Boc ODD1 and Boc ODD2 were consistent with the difference with the PRO/NAP value,indicating that Boc ODD1 and Boc ODD2 may play roles in the conversion of NAP to PRO.(4)Subcellular localization plays an important role in the systematic study of plant growth,development and gene function.By constructing GFP fusion protein and transfecting into tobacco,Boc ODD1 and Boc ODD2 were found to localized in cell membrane and nucleus.(5)To identify the function of BocODD1 and BocODD2,overexpression and RNAi interference vector were constructed and transformed into Chinses Kale by Agrobacterium-mediated method.The expression level of Boc ODD1/2 in transgenic plants of overexpression Boc ODD1/2 vector is upregulated,and down-regulate in RNAi transgenic plants,which cosistant with the PRO/NAP value and the contents of PRO glucosinolate,indicated that Boc ODD1 and Boc ODD2 simultaneously regulate the conversion of 3-butenyl glucoside to 2-hydroxy-3-butenyl glucosinolate.(6)Cloned the promoter of BocODD1/2 from Shengzhong and Tanshanzhonghua,obtained 1581 bp upstream segment of Boc ODD1 and 1403 bp upstream segment of Boc ODD2,the sequence from two varities have no difference,but all have MYB binding sites.The luciferase reporter system vector was constructed to transfect into tobacco and detected the luciferase activity.The luciferase activity of MYB28 co-transformed Boc ODD1 promoter into tobacco is 1.9 times that of the control,and the luciferase activity of the MYB28 co-transformed Boc ODD2 promoter was 3.3 times that of the control,indicating that MYB28 can active the promoter of Boc ODD1/2 and play an important role in positive regulate the expression of Boc ODD1/2 and the conversion value of NAP to PRO.