Van de Cappelle Elke, Plovie Eva, Kyndt Tina, Grunewald Wim, Cannoot Bernard, Gheysen Godelieve
Molecular Biotechnology Department, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium.
Plant Biotechnol J. 2008 Oct;6(8):749-57. doi: 10.1111/j.1467-7652.2008.00355.x. Epub 2008 Jun 11.
The activity of the Arabidopsis thaliana cyclin-dependent kinase AtCDKA;1 is important throughout G(1)/S and G(2)/M transitions and guarantees the progression of the cell cycle. Inhibitor studies have shown that activation of the cell cycle is important for the development of nematode feeding sites. The aim of this study was to silence the expression of the AtCDKA;1 gene in nematode feeding sites to interfere with their development. Therefore, sense and antisense constructs were made for the AtCDKA;1 gene and fused to a nematode-inducible promoter which was activated in nematode feeding sites at an earlier time point than AtCDKA;1. Two transgenic A. thaliana lines (S266 and S306) containing inverted repeats of the AtCDKA;1 gene and with reduced AtCDKA;1 expression in seedlings and galls were analysed in more detail. When the lines were infected with the root-knot nematode Meloidogyne incognita, significantly fewer galls and egg masses developed on the roots of the transgenic than wild-type plants. Infection of the AtCDKA;1-silenced lines with Heterodera schachtii resulted in significantly fewer cysts compared with controls. The S266 and S306 lines showed no phenotypic aberrations in root morphology, and analysis at different time points after infection demonstrated that the number of penetrating nematodes was the same, but fewer nematodes developed to maturity in the silenced lines. In conclusion, our results demonstrate that silencing of CDKA;1 can be used as a strategy to produce transgenic plants less susceptible to plant-parasitic nematodes.
拟南芥细胞周期蛋白依赖性激酶AtCDKA;1的活性在整个G(1)/S和G(2)/M转变过程中都很重要，并确保细胞周期的进程。抑制剂研究表明，细胞周期的激活对线虫取食位点的发育很重要。本研究的目的是使线虫取食位点中AtCDKA;1基因的表达沉默，以干扰其发育。因此，构建了AtCDKA;1基因的正义和反义构建体，并与一个线虫诱导型启动子融合，该启动子在比AtCDKA;1更早的时间点在线虫取食位点被激活。对两个含有AtCDKA;1基因反向重复序列且幼苗和虫瘿中AtCDKA;1表达降低的转基因拟南芥品系（S266和S306）进行了更详细的分析。当这些品系被根结线虫南方根结线虫感染时，转基因植物根部形成的虫瘿和卵块明显少于野生型植物。与对照相比，用甜菜孢囊线虫感染AtCDKA;1沉默品系导致形成的孢囊明显减少。S266和S306品系在根形态上没有表现出表型异常，感染后不同时间点的分析表明，侵入的线虫数量相同，但在沉默品系中发育成熟的线虫较少。总之，我们的结果表明，沉默CDKA;1可以作为一种策略来培育对植物寄生线虫更不易感的转基因植物。
