摘要
通过挖掘辣椒疫霉中的外泌蛋白激发子elicitins的基因序列,分析其转录特征,克隆基因序列并进行瞬时表达,以阐明其在辣椒疫霉生长发育和侵染过程中的作用。本研究利用转录组测序结果和致病疫霉INF1序列作为比对序列,对辣椒疫霉基因组数据库进行elicitins序列挖掘。采用RT-PCR方法分析elicitins基因在辣椒疫霉生长发育(菌丝、游动孢子囊、游动孢子、萌发的休止孢)和侵染寄主阶段(本氏烟灌根1.5、3、6、12、24、36和72 h后)的转录水平。克隆elicitins基因序列,并与其他卵菌的elicitins进行序列比对,分析进化关系。在本氏烟上进行elicitins的瞬时表达分析。结果表明:经生物信息学分析获得14个含有信号肽编码序列的elicitins基因全长序列。新发现6个elicitins基因在辣椒疫霉生长发育和侵染寄主阶段差异表达。对这6个和之前报道的5个elicitins基因进行全长克隆,能够克隆出的10个elicitins都属于酸性elicitins,聚类分析可将其分在第一和第三聚类组。异源表达发现2个elicitins能够激发本氏烟产生过敏反应。本研究结果不仅为阐明elicitins在辣椒疫霉生长发育和侵染寄主过程中的作用提供了重要数据,也为植物抗疫病的基因工程提供了科学依据。
Elicitins are a family of small elicitor proteins secreted by species of the genera Pythium and Phytophthora inducing a hypersensitive reaction and systemic acquired resistance in Nicotiana and Solanum spp. within the Solanaceae family and Brassica spp. within the Cruciferae family. The paper reports the mining of elicitin gene of Phytophthora capsici,their transcription levels at different stages,their cloning and transient expression in plants. These results contribute to better understanding the roles of elicitins in the interactions between P. capsici and its host plants as well as in the developmental stages. Elicitin candidate genes were mined from the recently released genome database of P. capsici using Phytophthora infestans INF1 and our Illumina transcriptome data as tBLASTx queries（ an expected value cut-off of 1e-50）. The transcriptional level of the predicted elicitin genes was investigated using RT-PCR technique during the development stages（ mycelium,sporangium,zoospore and germinating cyst） of P. capsici and its infection phases of Nicotiana benthamiana roots（ 1. 5,3,6,12,24,36,72 h post-inoculation）. Primer pairs specific to the genes were employed to amplify their full lengths using high-fidelity DNA polymerase. Phylogenetic tree was constructed based on multiple sequence alignments of them with other Phytophthora and Pythium spp. elicitins. In the end,these elicitin genes were transiently expressed in N. benthamiana leaves using Potato virus X（ PVX） agroinfiltration assay. Blasting search yielded 26 P. capsici sequences highly similar to P. infestans INF1 and our previous elicitin data from transcriptome sequencing. Only 14 elicitin candidate genes with putative signal peptide-encoding sequences and full lengths were obtained. Transcriptional analysis discovered 6 novel ones from these elicitin genes that were differentially expressed during the developmental and infection stages of P. capsici. The transcriptional profiling showed major differences between the infection stages,which can classify the genes＇ expression into 3 different types： low or no transcripts detected at the early infection stages,but up-regulation at later stages; constant expression at designated time-points; weak expression at the early infection stages,and gradually increasing along with the proceeding time-points. The fulllengths of 10 out of 11 elicitin genes（ including 6 ones found in this study and 5 previously reported） were successfully cloned,and amino sequences deducted from these genes showed that they are acidic elicitins. Phylogenetic analysis displayed that these 10 elicitins were clustered into class Ⅲ andⅠ. Ectopic expression indicated that 2 elicitins genes（ Pc508763 and Pc508770） can trigger the hypersensitive reaction in N. benthamiana. The results laid an essential foundation not only for elucidating the interaction mechanisms between P. capsici and its hosts but for future engineering of plant Phytophthora disease resistance.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2014年第6期28-36,共9页
Journal of Nanjing Agricultural University
基金
江苏省大学生实践创新训练计划项目(2012JSSPITP1356)
国家大学生创新创业训练计划项目(201211117063)
扬州大学大学生创新创业训练计划项目(b13194)
江苏省高校自然科学研究项目(13KJB210009)
江苏省自然科学基金项目(BK2011443)
国家自然科学基金项目(31101395)
