| Wheat is one of the most important crops, its yield and quality are directly related to the food security and living standards of the people. Transgenic technology has gradually become one of the effective ways to improve the quality of wheat.Lipoxygenase(LOX) can catalyze oxidation of unsaturated fatty acid in wheat(Triticum aestivum) seed, producing hydroperoxide during storage, which lead to decrease of seed viability and nutritional quality. Therefore, depressing LOX activity is an important mission for wheat genetic improvement.This study analysised the storability of seeds from wheat Lipoxygenase gene RNAi transgenic lines, aimed to get transgenic lines with significant reduction of lipoxygenase activity and increased seed storage-tolerance. The research content and results are as follows:(1)The transgene Loxi(Lox gene RNAi construct) and Bar gene of TF7 and TF8 progenies from transgenic lines(Xinong 889, Yanmai 8911 and Shanyou 225)was detected by PCR analysis and Basta Testing, and the ipoxygenase activity of TF7 transgenic lines were also tested. The results showed that all of the experimental materials used showed no basta resistance. For Xinong 889 and Yanmai 8911, all the TF7 and TF8 transgenic lines tested had the Loxi gene, while for Shanyou 225, 2 of TF7 and 1 of TF8 transgenic lines had no Loxi gene. All of these results demonstrate that after selection for several generations, all transgenic lines had no Bar gene, and Loxi gene was steadily inherited in all transgenic lines except the transgenic lines of Shanyou 225. Lipoxygenase activity test results showed that compared with their parents Xinong 889, Yanmai 8911 and shan 889, transgenic lines showed lower lipoxygenase activity, which was 9.97% ~ 9.97%, 24.35% ~ 90.67% and 26.31% ~90.67% of their parents, respectively. This result showed that exogenous Loxi can effectively inhibit lipoxygenase activity.(2)Using 6 TF7 transgenic lines of wheat(T. aestivum) from 3 populations(2transgenic lines from each population) as material, the expression of three Lox gene in seeds was detected by semi-quantitative RT-PCR. The results showed that the exogenous Loxi specifically suppressed the expression of TaLox1 gene in transgenic seeds at different level, in which the expression of TaLox1 gene was suppressed significantly in two transgenic lines of Shanyou225. The expression of TaLox2 and TaLox3 remained unaffected in all transgenic lines. it showed that the Loxi Can specifically inhibit TaLox1 which eventually lead to the decrease of lipoxygenase activity in seeds.(3)The seeds from TF7 transgenic lines of Xinong 889, Yanmai 8911 and Shanyou 225, which with Loxi and low elipoxygenase activity, were treated by artificial accelerated aging for 7 d and 14 d, then the lipoxygenase activity,germination rate, and shoot length and fatty acid composition were detected. The results showed that after artificial aging treatment, the lipoxygenase activity of all the material except SY7-1 increased significantly, while the transgenic lines increased less than their parents. The germination percentage and shoot length declined both in transgenic lines and their parents, while it decreased less in transgenic lines than their parents. GC-MS(Gas chromatography-mass spectrometer) analysis revealed that reduction of unsaturated fatty acid level in transgenic seeds during storage was lower when compared with that of non-transgenic seeds.All of these results indicated that Lipoxygenase gene RNAi can effectively suppress the expression of TaLox1 gene, reduced the LOX activity of transgenic seeds, and enhanced seed germination and storage-tolerance than that of nontransgenic seeds. These transgenic wheat lines with low lipoxygenase activity can be used as marterials or new germplasm in wheat breeding program. |
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