论文部分内容阅读
对20个百合种质资源的组培苗进行尖孢镰刀菌百合专化型(Fusarium oxysporum f.sp.lilii)室内接种鉴定;以筛选出有较好抗性的大花卷丹(Lilium leichtlinii var.maximowivzii Baker)为材料,以镰刀菌诱导12和24 h的作为处理组,无菌水处理作为对照组,采用SMART技术构建了百合经镰刀菌诱导后的抑制差减正、反向两个SSH文库。在正向文库中选择了4个抗病相关的ESTs,用半定量RT-PCR分析这些ESTs的表达情况。经接种鉴定,20个百合种质资源中,对百合镰刀菌表现高抗的有3个,中抗9个,感病8个;SSH文库ESTs片段大小为200~2 000 bp。依据斑点杂交结果,从正向SSH文库中挑取50个单克隆进行测序和比对分析,得到6种共10条抗病相关EST。通过半定量RT-PCR对其中的4条抗病相关ESTs:丝氨酸/苏氨酸蛋白激酶(serine/threonine protein kinase,S/TPK)、谷胱甘肽S–转移酶(glutathione S-transferase,GST)、过氧化物酶(peroxidase,POD)和亲环素(cyclophilin,CYP)的表达情况进行了分析,证明它们在一定程度上都受百合镰刀菌诱导而表现上调表达,推测其可能参与了百合对镰刀菌枯萎病的抗病过程。
Twenty Lilium germplasm resources of tissue culture seedlings were inoculated with Fusarium oxysporum f.sp.lilii (Fusarium oxysporum f.sp.lilii) in vitro identification; to screen out a good resistance of large flowers curl (Lilium leichtlinii var. maximowivzii Baker) were used as the treatment group, Fusarium-induced 12 and 24 h treatment groups, sterile water treatment as the control group, SMART technology was used to construct the Lily Fusarium inhibition-subtracted positive and negative two SSH library . Four disease-related ESTs were selected in the forward library and the expression of these ESTs was analyzed by semi-quantitative RT-PCR. After inoculation, 3 out of 20 lily germplasm resources showed high resistance to Fusarium lactuca, 9 were moderately resistant and 8 were susceptible. The ESTs fragments of SSH library ranged from 200 to 2 000 bp. According to the results of dot blot hybridization, 50 clones were picked from the forward SSH library for sequencing and comparison analysis, and 6 kinds of ESTs were obtained. Four disease-related ESTs were identified by semi-quantitative RT-PCR: serine / threonine protein kinase (S / TPK), glutathione S-transferase (GST ), Peroxidase (POD) and cyclophilin (CYP) were analyzed, and they were all confirmed to be upregulated by Fusarium species in a certain extent, suggesting that they may be involved in the synthesis of lily Fusarium wilt disease resistance process.