Oomen Ronald J F J, Tzitzikas Emmanouil N, Bakx Edwin J, Straatman-Engelen Irma, Bush Maxwell S, McCann Maureen C, Schols Henk A, Visser Richard G F, Vincken Jean-Paul
Graduate School Experimental Plant Sciences, Laboratory of Plant Breeding, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands.
Phytochemistry. 2004 Mar;65(5):535-46. doi: 10.1016/j.phytochem.2003.12.019.
Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis.
通过筛选由发育中的块茎构建的cDNA文库,分离出了四个马铃薯纤维素合酶(CesA)同源物(StCesA1、2、3和4)。基于序列比较以及所有四个马铃薯cDNA均从该单一cDNA文库中分离这一事实,所有四个StCesA克隆可能在初生细胞壁生物合成中发挥作用。构建了几个载体,以调节马铃薯植株中的纤维素水平,其中利用颗粒结合淀粉合酶启动子将修饰作用靶向到块茎。通过完整cDNA的正义(SE-StCesA3)和反义(AS-StCesA3)表达,使用StCesA3来上调和下调纤维素水平。此外,所有四个马铃薯纤维素合酶基因的类特异性区域(CSR)用于相应CesA基因(csr1、2、3和4)的特异性下调(反义)。没有一个转化体表现出明显的发育表型。通过傅里叶变换红外光谱(FTIR)和探索性主成分分析(PCA)筛选块茎切片中细胞壁结构的变化,并对那些与野生型植株有差异的植株进行纤维素含量和单糖组成分析。获得了几个主要纤维素水平降低的转基因株系。这些结果表明,马铃薯块茎中的纤维素含量可以降低到野生型水平的40%,而不影响植株的正常发育,并且仅基于CSR的载体具有特异性且足以下调纤维素生物合成。