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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。
By digging the gene sequence of elicitins, the exoprotein elicitor in Phytophthora capsici, the transcriptional characteristics were analyzed, and the gene sequences were cloned and transiently expressed to clarify its role in the growth and development of Phytophthora capsici. In this study, the sequence of the transcriptome and the INF1 sequence of Phytophthora infestans were used as alignment sequences to search for the elicitins of Phytophthora capsici genomic databases. The expression of elicitins gene in Phytophthora capsici (mycelium, zoospore, zoospore and apricot) was analyzed by RT-PCR and infected hosts (1.5, 24, 36 and 72 h). The elicitins gene sequence was cloned and aligned with elicitins from other oomycetes to analyze the evolutionary relationships. Transient expression analysis of elicitins was performed on N. benthamiana. The results showed that the full-length sequence of elicitins containing 14 signal peptide coding sequences was obtained by bioinformatics analysis. Six new elicitins genes were found to be differentially expressed at the host stage of Phytophthora capsici growth and invasion. Of the 6 previously reported 5 elicitins genes cloned in full length, all 10 elicitins that can be cloned are acidic elicitins, and cluster analysis can be grouped into the first and third cluster groups. Heterologous expression of two elicitins found to stimulate Benny smoke allergic reactions. The results of this study not only provide important data for elucidating the role of elicitins in the growth and host infection of Phytophthora capsici, but also provide a scientific basis for genetic engineering of plant disease resistance.