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Copyright of Agricultural Science & Technology is the property of Hunan Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Agricultural Science & Technology is the property of Hunan Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

[Objective] This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering (DAF) as well as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradually increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter (ODP) in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expression decline was well coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely controlled by the napin promoter. [ABSTRACT FROM AUTHOR]

The genetically modified high-oleic rapeseed(Brassica napus L.) line W-4 was obtained by transforming a binary vector which harbored an inverted repeat expression cassette of fad2 gene into the rapeseed cultivar Westar. The transformation was mediated by Agrobacterium. The flanking sequences to both the left and right borders of T-DNA insertion site were amplified by thermal asymmetric interlaced PCR(TAIL-PCR) from the genomic DNA of the transgenic rapeseed line W-4. The flanking sequences to the right border was 290 bp in length and the nucleotide composition was 31.27% for G+C content while 68.73% for A+T content. The flanking sequence to the left border was 365 bp in length and the G +C content was 32.6% and the A+T content was 67.4%, indicating that the T-DNA was integrated in the A/T-rich region. Further more, sequence alignment analysis showed a deletion of 62 bp including the right border of pCNFIRnos and the integration of the whole left border except a change of G to A. That was to say, the integration of the T-DNA in the transgenic line W-4 not involved in the vector sequences. Based on both flanking sequences as well as the left and right borders of the T-DNA sequences,two pairs of specific primers TLF/TLR and TRF/TRR were designed. Using the primers the event-specific PCR detection method for transgenic rapeseed line W-4 was established. By the PCR, two fragments of 485 and 405 bp were amplified from the W-4 genomic DNA as expected, while no products were amplified from the genomic DNA of other transgenic rapeseed lines and non-transgenic rapeseed line.And by the PCR it is possible to detect the W-4 genomic DNA from a mixed sample of genomic DNA. The limit of the detection for the qualitative PCR assay was 0.1%. The method developed in this work is highly specific, sensitive and suitable for event-specific detection of the transgenic rapeseed line W-4. [ABSTRACT FROM AUTHOR]