Klink Vincent P, Kim Kyung-Hwan, Martins Veronica, Macdonald Margaret H, Beard Hunter S, Alkharouf Nadim W, Lee Seong-Kon, Park Soo-Chul, Matthews Benjamin F
Department of Biological Sciences, Mississippi State University, Harned Hall, Rm 310, Mississippi State, MS 39762, USA.
Planta. 2009 Jun;230(1):53-71. doi: 10.1007/s00425-009-0926-2. Epub 2009 Apr 4.
Host-mediated (hm) expression of parasite genes as tandem inverted repeats was investigated as a means to abrogate the formation of mature Heterodera glycines (soybean cyst nematode) female cysts during its infection of Glycine max (soybean). A Gateway-compatible hm plant transformation system was developed specifically for these experiments in G. max. Three steps then were taken to identify H. glycines candidate genes. First, a pool of 150 highly conserved H. glycines homologs of genes having lethal mutant phenotypes or phenocopies from the free living nematode Caenorhabditis elegans were identified. Second, annotation of those 150 genes on the Affymetrix soybean GeneChip allowed for the identification of a subset of 131 genes whose expression could be monitored during the parasitic phase of the H. glycines life cycle. Third, a microarray analyses identified a core set of 32 genes with induced expression (>2.0-fold, log base 2) during the parasitic stages of infection. H. glycines homologs of small ribosomal protein 3a and 4 (Hg-rps-3a [accession number CB379877] and Hg-rps-4 [accession number CB278739]), synaptobrevin (Hg-snb-1 [accession number BF014436]) and a spliceosomal SR protein (Hg-spk-1 [accession number BI451523.1]) were tested for functionality in hm expression studies. Effects on H. glycines development were observed 8 days after infection. Experiments demonstrated that 81-93% fewer females developed on transgenic roots containing the genes engineered as tandem inverted repeats. The effect resembles RNA interference. The methodology has been used here as an alternative approach to engineer resistance to H. glycines.
研究了宿主介导的（hm）寄生虫基因串联反向重复表达，以此作为一种手段来消除大豆孢囊线虫（Heterodera glycines）在感染大豆（Glycine max）过程中成熟雌虫孢囊的形成。专门为这些大豆实验开发了一种与Gateway兼容的hm植物转化系统。然后采取了三个步骤来鉴定大豆孢囊线虫的候选基因。首先，从自由生活的线虫秀丽隐杆线虫（Caenorhabditis elegans）中鉴定出150个具有致死突变表型或拟表型的高度保守的大豆孢囊线虫同源基因库。其次，对Affymetrix大豆基因芯片上的这150个基因进行注释，从而鉴定出131个基因的子集，其表达可在大豆孢囊线虫生命周期的寄生阶段进行监测。第三，微阵列分析确定了一组核心的32个基因，这些基因在感染的寄生阶段有诱导表达（>2.0倍，以2为底的对数）。在hm表达研究中测试了小核糖体蛋白3a和4（Hg-rps-3a [登录号CB379877]和Hg-rps-4 [登录号CB278739]）、突触融合蛋白（Hg-snb-1 [登录号BF014436]）和剪接体SR蛋白（Hg-spk-1 [登录号BI451523.1]）的大豆孢囊线虫同源基因的功能。感染8天后观察到对大豆孢囊线虫发育的影响。实验表明，在含有经工程改造为串联反向重复序列的基因的转基因根上发育的雌虫减少了81-93%。这种效应类似于RNA干扰。该方法已在此处用作培育抗大豆孢囊线虫的替代方法。
