两个紧密连锁的小麦苯丙氨酸解氨酶基因的分离与鉴定

Isolation and Characterization of Two Closel y Linked Phenylalanine Ammonia-Lyase Genes from Wheat

利用一个小麦苯丙氨酸解氨酶基因PCR片段为探针 ,从小麦核DNA基因库中筛选出一个阳性噬菌体克隆 ,该克隆含有两个高度同源、紧密连锁、转录方向相同的小麦苯丙氨酸解氨酶基因PAL1与PAL2 ,它们之间的核酸序列同源性为 93% ,相距约 7kb。利用PAL1特异片段进行Southern分析 ,表明该基因在小麦基因组中具有多个拷贝。Northern杂交表明 ,经秆锈菌接种诱导 ,苯丙氨酸解氨酶基因在一对小麦抗 感近等基因系中差异表达 :抗病等基因系中国春 Sr11携带与接种菌无毒性基因P11相对应的抗病基因Sr11,在接种 4d后开始诱导表达 ,8d后表达量更高 ;而缺少抗病基因的感病系中国春 sr11接种 6d后才开始表达 ,8d后的表达量与抗病系中 6d时相当。用秆锈菌诱导物和几丁质寡聚物处理小麦悬浮细胞 ,均可在 2h内激活苯丙氨酸解氨酶基因表达 ,但真菌诱导物在早期的诱导活性显著高于几丁质寡聚物。

Two closely linked,highly homologous phenylalanin e ammonia-lyase (PAL) genes located in one phage clone,PAL1 a nd PAL2,were isolated from a wheat genomic library by using a po lymerase chain reaction (PCR) fragment of a wheat PAL gene as probe.The two PAL genes were located approximately from 7 kb apart and displayed 93% ident ity with the same orientation.Southern blot analysis with a PAL1 specific fragment as probe showed the presence of a multiple gene f amily of PAL in wheat.Northern analyses demonstrated a differential expressi on of PAL in two Chinese Spring near-isogenic lines upon infection with ste m rust fungus Puccinia graminis.In the resistant Chinese Spring Sr11 isogeni c line with the resistance gene Sr11 that is known t o interact with the avirulence gene P11 from the ste m rust fungus,an induced expression of PAL was o bserved 4 days post inoculation (d.p.i.),and a massive induction was evident 8 d .p.i.By contrast,in the susceptible Chinese Spring sr11 line lacking the resista nce gene,induction of PAL was seen 6 d.p.i.and the expression level at 8 d.p .i.was similar to that observed in the resistant line at 6 d.p.i.In wheat suspen sion culture cells,treatment with either an elicitor isolated from the stem rust fungus or chitin oligomers could activate PAL gene expression within 2 h.Th e fungal elicitor appeared to be more active in early activation of the PAL gene than were chitin oligomers.These results demonstrated that wheat PAL pl ayed an inportant role in the induced resistance response upon infection with st em rust fungus at transcription level.