| The content of erucic acid is one of the important factors that affect the quality of rapeseed oil. Low erucic acid rapeseed (LEAR) has already been developed using conventional breeding techniques but due to high erucic acid rapeseed being still planted, the erucic acid content in the commercial LEAR oil tends to be elevated. To ensure the LEAR quality and to broaden the spectrum of hybrid parents, it is attracting to silence the genes related to eucic acid biosynthesis and to develop a rapeseed line in which the low erucic acid trait is dominant. In this study, the distribution of the natural mutants of low erucic acid was investigated among 30 Chinese LEAR varieties; the molecular mechanism of LEA was surveyed with the focus on the effect of two-base deletion at 1422-1423th of FAE1.2; the related genes on the erucic acid synthesis were silenced separately with the aim to develop transgenic and dominant LEAR.The main results are as follows:1. The natural mutants of FAE1 in LEAR:FAE1 genes were cloned and sequenced from 30 LEARs including the first LEAR "Oro", and from the HEAR "Zhongyou821" and three types of LEAR mutation were distinguished. The heterologous expression of FAE1 in yeast showed that eC2 resulted in the absence of the condensing enzyme and led to the failure to produce C22:0 and C22:1. Moreover, KCR and ECR were still coordinately regulated in LEAR, but not in HEAR, indicating that the expression of two genes was tightly linked.2. Silencing the genes involved in the erucic acid synthesis:The anti-sense and ihpRNA interference vectors targeting FAE1, ECR, KCR and HCD were constructed and the number of positive transformants was totally 109. The average erucic acid content of transgenic T2 seed for FAE1 and HCD anti-sense inhibition was 34.36% and 34.33% respectively, decreased by 8.6% and 8.63% compared with the control(p<0.01); the line aFAE1189B134 had the lowest erucic acid content(13.33%). Among ihpRNA transgenic plants, the average erucic acid content of transgenic lines for FAE1 was 34.29%(p<0.01) with the minimum content being 24.56%(iFAE1229B75). The average erucic acid content of iKCR lines dropped to 32.03% with the minimum content being 16.09%. The average erucic acid content of iHCD lines was 33.76%(p<0.01) and line iHCD368 had the lowest erucic acid content of 8.53%. For iECR lines, the erucic acid was 39.87%(p>0.05). RNAi for genes FAE1, KCR, ECR and HCD affected the erucic acid synthesis in rapeseed seeds, indicating that these genes played indispensable roles in the erucic acid synthesis process and could be used as target genes in the breeding of transgenic LEAR.3. The selection of transgenic LEAR:The average erucic acid content of pl21NapDsfae1D500, p121NapDsfae1D800 and p121NapDsfae1D1500 dropped to 28.03%(p<0.01),18.23%(p<0.01) and to 31.0%(p<0.05) respectively. The copy number was confirmed by Southern blot analysis. The average relative expression of FAE1 was declined by more than 60%(p<0.01) and i800-821059A17 declined by 97% which was the maximum reduced amount among all transgenic lines. The expression levels of KCR and ECR were significantly reduced than the control(p<0.01). The expression of FAE1 protein was week in seed of the transgenic lines i800-821023A16, i800-821059A17 and i800-821068A18.The erucic acid content in T2 and T3 seeds for line i800-821068A18 was 1.58%,3.11% respectively, and this indicated that low-erucic-acid trait was stably hereditable in this transgenic line. The oil content of T2 seeds for transgenic lines i800-821068A18 was 35.18%,6.43% lower than the control; the glucosinolate content was 108.06μmol/g and the protein content was 24%, increased by 4.05%. The transcript levels of the genes involved in fatty acid synthesis were disturbed in various degrees. The erucic acid content in hybrid seed from reciprocal cross between i800-821068A18 and HEAR was 5.47% and 2.95% respectively, meeting the low erucic acid standard. |
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