Wroblewski Tadeusz, Piskurewicz Urszula, Tomczak Anna, Ochoa Oswaldo, Michelmore Richard W
The Genome Center, University of California in Davis, Davis, CA 95616, USA.
Plant J. 2007 Sep;51(5):803-18. doi: 10.1111/j.1365-313X.2007.03182.x. Epub 2007 Jun 22.
The RGC2 gene cluster in lettuce (Lactuca sativa) is one of the largest known families of genes encoding nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins. One of its members, RGC2B, encodes Dm3 which determines resistance to downy mildew caused by the oomycete Bremia lactucae carrying the cognate avirulence gene, Avr3. We developed an efficient strategy for analysis of this large family of low expressed genes using post-transcriptional gene silencing (PTGS). We transformed lettuce cv. Diana (carrying Dm3) using chimeric gene constructs designed to simultaneously silence RGC2B and the GUS reporter gene via the production of interfering hairpin RNA (ihpRNA). Transient assays of GUS expression in leaves accurately predicted silencing of both genes and were subsequently used to assay silencing in transgenic T(1) plants and their offspring. Levels of mRNA were reduced not only for RGC2B but also for all seven diverse RGC2 family members tested. We then used the same strategy to show that the resistance specificity encoded by the genetically defined Dm18 locus in lettuce cv. Mariska is the result of two resistance specificities, only one of which was silenced by ihpRNA derived from RGC2B. Analysis of progeny from crosses between transgenic, silenced tester stocks and lettuce accessions carrying other resistance genes previously mapped to the RGC2 locus indicated that two additional resistance specificities to B. lactucae, Dm14 and Dm16, as well as resistance to lettuce root aphid (Pemphigus bursarius L.), Ra, are encoded by RGC2 family members.
生菜（Lactuca sativa）中的RGC2基因簇是已知最大的编码核苷酸结合位点富含亮氨酸重复序列（NBS-LRR）蛋白的基因家族之一。其成员之一RGC2B编码Dm3，Dm3决定了对携带同源无毒基因Avr3的卵菌莴苣盘梗霉引起的霜霉病的抗性。我们开发了一种利用转录后基因沉默（PTGS）分析这个低表达基因大家族的有效策略。我们使用嵌合基因构建体转化生菜品种Diana（携带Dm3），该构建体设计通过产生干扰性发夹RNA（ihpRNA）同时沉默RGC2B和GUS报告基因。叶片中GUS表达的瞬时分析准确预测了两个基因的沉默，随后用于分析转基因T(1)植株及其后代中的沉默情况。不仅RGC2B的mRNA水平降低了，而且所测试的所有七个不同RGC2家族成员的mRNA水平也降低了。然后我们使用相同的策略表明生菜品种Mariska中由遗传定义的Dm18位点编码的抗性特异性是两种抗性特异性的结果，其中只有一种被源自RGC2B的ihpRNA沉默。对转基因沉默测试品系与携带先前定位到RGC2位点的其他抗性基因的生菜种质之间杂交后代的分析表明，RGC2家族成员还编码了对莴苣盘梗霉另外两种抗性特异性Dm14和Dm16以及对生菜根蚜（Pemphigus bursarius L.）的抗性Ra。
