Regulation Of Erucic Acid Content In Brassica Carinata Seeds

Brassica carinata is a Brassica species with high tolerance to drought,blackleg disease and white rust.The rich erucic acid in B.carinata seed oil is widely used for industrial products such as erucamid.The biosythesis of erucic acid in plants is regulated mainly by three kinds of genes,which are fatty acid elongase(FAE1),fatty acid desaturase(FAD2)and lysophosphatidic acid acyltransferase(LPAAT).In this study,endogenous LPAAT-RNAi structure was added to the plasmid vecter pWatergate-napin-FAD2i-LdLPAAT-KCS,which was constructed to regulate boisythesis of erucic in developing seeds of.Brassica carinata.Primer of NPTIIgene was designed to confirm transgenetic lines in TO.Part of T1 seeds received from 40 TO generation lines containing fragment of NPTIIgene were directly sowed and transgene identification by PCR with NPTII primer and LdLPAAT primer.The other part was analyzed by gas chromatography for erucic acid content determination.Transgenic identification was performed on 40 T1 lines with high erucic acid content received from 36 TO lines.The results were summarize as follows:(1)Analyzation of fatty acid content of wild type type B.carinata seed oil;Ten wild-type mustard oils were selected for fatty acid composition and erucic acid content analysis,and a total of 9 fatty acid components were detected.The average content of erucic acid,linoleic acid and linolenic acid in wild type B.carinata seed oil was 40.466%,16.689%,and 13.871%,respectively;Analyzation of erucic acid content in seeds of 40 T1 lines:(2)Four T1 lines,i.e.,143-1-6,59-5-1,59-2-2 and 59-2-3,contain erucic acid higher than 48%,and their erucic acid content were 48.582%,48.019%,48.413%and 50.111%,respectively.Line59-2-3 contains 50.111%erucic acid,12.392%oleic acid,12.704%linolenic acid,7.064%linolenic acid.Compared to the wild type,the erucic acid was increased by 23.83%,the oleic acid content decreased by 9.47%,the linoleic acid decreased by 24.69%,and linolenic acid decreased by 49.30%;(3)17 T1 lines contain erucic acid between 45%and 48%.The erucic acid in line 6-3-4 seed was 46.498%,with 10.386%oleic acid,17.892%linoleic acid,and 12.905%linolenic acid.The oleic acid content was decreased by 24.13%,linoleic acid increased by 6.05%,linolenic acid decreased by 7.38%,and erucic acid increased by 14.90%,compared with the wild type;(4)19 T1 lines contain erucic acid content lower than 45%.Among them,line 2-9-12 contains 43.491%erucic acid,8.485%oleic acid,22.938%linoleic acid,11.592%linolenic acid.The linolenic acid was 38.01%.The linoleic acid content was increased by 35.96%,the linolenic acid content decreased by 16.80%,and the erucic acid content increased by 7.47%,compared with the wild type;(5)Transgenic identification of the above 40 strains,the PCR results showed that the four lines containing more than 48%erucic acid could amplify the NPTII and LdLPAAT target fragments.All of the 17 lines with erucic acid content ranging from 45%to 48%containing the fragments of NPTII,LdLPAAT fragments could be amplified except line 22-1-1,22-3-2,48-1-1,59-3-6 and 52-1-1.Among the lines with erucic acid content ranging from 43%to 45%,NPTII target fragments could be identified except line 17-3-7 and 142-3-1,LdLPAAT target fragments could be amplified with the exception of linel 76-1-4,51-1-1,6-4-2 and 47-1-1;(6)Among the 38 transgenic lines that could identify the NPTII target fragment,except for strains 59-2-3,the linoleic acid content of the remaining 36 strains was slightly hgher than that of the wild type,and the linolenic acid content were all lower than wild-type except line strain 5-2-5,they.This result indicates that FAD2i did not achieve the expected interference in these lines.