Plant Breeding and Acclimatization Institute, Radzikow, 05-870 Blonie, Poland.
J Exp Bot. 2010 Jun;61(6):1839-51. doi: 10.1093/jxb/erq052. Epub 2010 Mar 24.
Stable RNA interference-based technology was used to silence the expression of the HvCKX1 gene in barley and the TaCKX1 gene in wheat and triticale. The silencing cassettes containing the fragments of these genes in the sense and antisense orientations were cloned into the pMCG161 binary vector and used for Agrobacterium-based transformation. Out of the five cultivars representing the three studied species, transgenic plants were obtained from one barley cultivar Golden Promise, one wheat cultivar Kontesa, and one triticale cultivar Wanad. Almost 80% of 52 regenerated lines of Golden Promise exhibited significantly decreased cytokinin oxidase/dehydrogenase (CKX) enzyme activity in bulked samples of their T(1) roots. There was a positive correlation between the enzyme activity and the plant productivity, expressed as the yield, the number of seeds per plant, and the 1000 grain weight. Additionally, these traits were associated with a greater root mass. Lower CKX activity led to a higher plant yield and root weight. This higher plant productivity and altered plant architecture were maintained in a population of segregating T(1) plants. The levels of HvCKX1 transcript accumulation were measured in various tissues of Golden Promise and Scarlett non-transgenic barley plants in order to choose the most appropriate plant organs to study the expression and/or silencing of the gene in those transgenic lines. The highest levels of the HvCKX1 transcript were detected in spikes 0 days after pollination (0 DAP), 7 DAP, and 14 DAP, and in the seedling roots. The analysis of HvCKX1 gene expression and CKX enzyme activity and the evaluation of the phenotype were performed in the progeny of seven selected transgenic T(1) lines. The relative expression of HvCKX1 measured in the spikes 0 DAP and 14 DAP, respectively, ranged from 0.52+/-0.04 to 1.15+/-0.26 and from 0.47+/-0.07 to 0.89+/-0.15. The lowest relative values were obtained for the enzyme activity in the spikes at 0 DAP, which ranged from 0.15+/-0.02 to 1.05+/-0.14 per single progeny plant. Based on these three values, the coefficient of HvCKX1 silencing in the spikes was estimated. Possible mechanisms leading to higher plant productivity via the silencing of HvCKX1 and a decrease in CKX enzyme activity are discussed.
采用稳定的 RNA 干扰技术沉默大麦中的 HvCKX1 基因和小麦和黑小麦中的 TaCKX1 基因的表达。含有这些基因片段的沉默盒在正义和反义方向上被克隆到 pMCG161 二元载体中，并用于农杆菌转化。在代表三个研究物种的五个品种中，从一个大麦品种 Golden Promise、一个小麦品种 Kontesa 和一个黑小麦品种 Wanad 中获得了转基因植物。在 Golden Promise 的 52 个再生系中，近 80%的系在其 T(1)根的混合样品中表现出明显降低的细胞分裂素氧化酶/脱氢酶 (CKX) 酶活性。酶活性与植物生产力之间存在正相关，以产量、每株种子数和千粒重表示。此外，这些特性与更大的根质量有关。较低的 CKX 活性导致更高的植物产量和根重。这种更高的植物生产力和改变的植物结构在分离的 T(1)植物群体中得以维持。在 Golden Promise 和非转基因 Scarlett 大麦的各种组织中测量了 HvCKX1 转录物的积累水平，以便选择最合适的植物器官来研究这些转基因系中基因的表达和/或沉默。在授粉后 0 天（0 DAP）、7 DAP 和 14 DAP 的穗和幼苗根中检测到 HvCKX1 转录物的最高水平。在七个选定的转基因 T(1)系的后代中进行了 HvCKX1 基因表达和 CKX 酶活性的分析以及表型的评估。分别在授粉后 0 天和 14 天的穗中测量的 HvCKX1 的相对表达范围为 0.52+/-0.04 至 1.15+/-0.26 和 0.47+/-0.07 至 0.89+/-0.15。在授粉后 0 天的穗中获得的相对值最低，每个单株后代的 CKX 酶活性范围为 0.15+/-0.02 至 1.05+/-0.14。基于这三个值，估计了穗中 HvCKX1 的沉默系数。讨论了通过沉默 HvCKX1 和降低 CKX 酶活性导致植物生产力提高的可能机制。
