Stable transgene expression and random gene silencing in wheat
Stable transgene expression and random gene silencing in wheat
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DOI：
10.1046/j.1467-7652.2003.00023.x
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发表时间：
2003-07-01
影响因子：
13.8
通讯作者：
Muthukrishnan, S
中科院分区：
文献类型：
--
作者：
Anand, A;Trick, HN;Muthukrishnan, S
利用玉米泛素启动子-内含子控制的小麦病程相关蛋白基因、几丁质酶基因和β-1，3-葡聚糖酶基因，采用基因枪方法转化春小麦Bobwhite。在T-0代中表达PR蛋白基因的24个原代转基因株系中有20个在T-1或T-2代中被沉默。选择两个明显遗传上相同的再生体用于进一步表征，所述再生体来自用几丁质酶和β-1，3-葡聚糖酶转基因组合共轰击的单个愈伤组织，但在转基因表达方面不同。在一个纯合系中，在T-3植物中观察到转基因沉默，而对于转基因位点纯合的另一个系稳定表达并将转基因遗传到至少T-4代。Southern印迹分析的基因组DNA从两个线使用的isopromomeric甲基化敏感的酶，MspI和Hpall，揭示了较高程度的甲基化的CCGG序列的线与沉默的转基因位点。通过逆转录-聚合酶链反应、北方印迹和西方印迹分析检测到转基因在具有较小程度甲基化的系中稳定表达，而具有较高水平CCGG甲基化的系在T-3代没有转基因表达。在胞苷类似物，5-azacyticline（azaC）的存在下，从沉默的植物种子发芽，没有导致这种表型的逆转。
Wheat genes for pathogenesis-related (PR-)proteins, chitinase and beta-1,3-glucanase, under the control of maize ubiquitin promoter-intron were used for transforming the spring wheat 'Bobwhite', using a biolistic approach. Twenty of the 24 primary transgenic lines expressing the PR-protein genes in the T-0 generation were silenced in either the T-1 or T-2 generations. Two apparently genetically identical regenerants arising from a single callus co-bombarded with chitinase and beta-1,3-glucanase transgene combinations, but differing in the expression of the transgenes were selected for further characterization. In one homozygous line, transgene silencing was observed in the T-3 plants, while the other line homozygous for the transgene loci stably expressed and inherited the transgenes to at least the T-4 generation. Southern blot analyses of genomic DNA from the two lines using the isoschizomeric methylation-sensitive enzymes, MspI and Hpall, revealed a higher degree of methylation of CCGG sequences in the line with the silenced transgene locus. Analysis by reverse transcriptase-polymerase chain reaction, Northern blotting and Western blotting detected stable expression of the transgenes in the line with a lesser extent of methylation, whereas the line with a higher level of CCGG methylation had no transgene expression by the T-3 generation. The germination of seeds from the silenced plants in the presence of a cytidine analogue, 5-azacyticline (azaC), did not lead to a reversion of this phenotype.