Tucker Mark L, Burke Aimee, Murphy Charles A, Thai Vanessa K, Ehrenfried Mindy L
Soybean Genomics and Improvement Laboratory, Agricultural Research Service, United States Department of Agriculture, Bldg 006, 10300 Baltimore Ave, Beltsville, MD 20705-2350, USA.
J Exp Bot. 2007;58(12):3395-406. doi: 10.1093/jxb/erm188. Epub 2007 Oct 4.
Changes in transcript accumulation for cell wall-modifying proteins were examined in excised soybean root pieces colonized by soybean cyst nematodes (SCN), Heterodera glycines, using RT-PCR and soybean Affymetrix GeneChips. Sequence-specific PCR primer pairs were prepared from sequence data for core sequences in the GenBank soybean database and consensus sequences derived from the assembly of soybean ESTs. In addition, to identify previously uncharacterized soybean transcripts, degenerate primers were prepared for conserved motifs in cellulases (endo-1,4-beta-glucanases, EGases) and polygalacturonases (PGs) and these were used to amplify segments of transcripts that were then extended with 3' and 5' RACE. Several novel EGase and PG transcripts were identified. Gene expression patterns were determined by real-time RT-PCR for 11 EGases, three expansins (EXPs), 14 PGs, two pectate lyases (PLs), and two xyloglucan endotransglucosylase/hydrolases (XTHs) in soybean roots inoculated with SCN, non-inoculated roots, serial dissections of root tips, leaf abscission zones, flowers, apical buds, and expanding leaves. A large number of genes associated with cell wall modifications are strongly up-regulated in root pieces colonized by SCN. However, in contrast to most of the transcripts for cell wall proteins, two XTH transcripts were specifically down-regulated in the colonized root pieces. Gene expression in serial dissections of root tips (0-2 mm, and 2-7 mm) and whole roots indicate that the SCN up-regulated genes are associated with a wide range of developmental processes in roots. Also of interest, many of the cDNAs examined were up-regulated in petiole abscission zones induced to abscise with ethylene.
利用逆转录聚合酶链式反应(RT-PCR)和大豆Affymetrix基因芯片,研究了大豆胞囊线虫(SCN,即大豆异皮线虫)侵染的大豆离体根段中细胞壁修饰蛋白转录物积累的变化。从GenBank大豆数据库中的核心序列数据以及由大豆表达序列标签(EST)组装得到的共有序列制备了序列特异性PCR引物对。此外,为了鉴定之前未被表征的大豆转录物,针对纤维素酶(内切-1,4-β-葡聚糖酶,EG酶)和多聚半乳糖醛酸酶(PG)中的保守基序制备了简并引物,并用于扩增转录物片段,然后通过3'和5'末端快速扩增(RACE)进行延伸。鉴定出了几种新的EG酶和PG转录物。通过实时RT-PCR测定了接种SCN的大豆根、未接种根、根尖连续切片、离层区、花、顶芽和正在伸展叶片中11种EG酶、3种扩张蛋白(EXP)、14种PG、2种果胶酸裂解酶(PL)和2种木葡聚糖内转糖基酶/水解酶(XTH)的基因表达模式。大量与细胞壁修饰相关的基因在被SCN侵染的根段中强烈上调。然而,与大多数细胞壁蛋白的转录物相反,两种XTH转录物在被侵染的根段中特异性下调。根尖(0 - 2毫米和2 - 7毫米)和整个根的连续切片中的基因表达表明,SCN上调的基因与根中广泛的发育过程相关。同样有趣的是,许多检测的cDNA在被乙烯诱导脱落的叶柄离层区中上调。