藜叶片响应出芽短梗霉菌侵染内参基因筛选及光合作用相关基因表达分析

Selection of reference genes and expression analysis of photosynthesis-related genes responding to Aureobasidium pullulans in Chenopodium album

【目的】筛选适合出芽短梗霉菌株PA-2侵染藜的稳定表达的最适内参基因,并进行PA-2侵染下藜光合作用相关基因的表达分析验证。【方法】分别以菌株PA-2孢子侵染0、1、3、5、7 d的藜叶片和正常生长藜的根、茎、叶为供试材料,通过qRT-PCR技术和Ct值评估3个候选内参基因GAPDH、β-ACTIN、β-TUBULIN的表达稳定性,并利用筛选出的内参基因,对藜光合作用相关基因(LFRN、MDH、PetC、psaA、CAB40、psaN、BHY和MO_(2))的表达水平进行定量分析。【结果】在菌株PA-2侵染藜不同时间点和不同组织的样本中,藜最适内参基因为GAPDH,可用于后续分析光合作用相关基因的表达水平。以GAPDH作为内参基因检测8个光合作用相关基因的表达水平,在不同侵染时间点,8个光合作用相关基因的表达量均出现不同程度的下调,除MDH基因外,其余基因均在5 d时达到最低水平。其中PctC、psaN、CAB40、BHY、MO_(2)基因在藜受侵染0~1 d中下调表达,在3 d达到较高水平,后期表达量下调;pasA先上调表达后下调表达;LFRN在受侵染后持续下调表达;MDH在受侵染后先显著下调,在3 d达到较低水平,后上调表达。在藜受侵染的不同组织中,8个基因在藜叶中均有大量表达,其中MDH基因在茎中大量表达,PctC和CAB40在根中表达量最低,除MDH基因外,其余7个基因在不同组织中的表达量高低排序为:叶>茎>根。【结论】本研究为后续利用qRT-PCR分析藜基因表达及研究PA-2致病机理奠定基础。

【Objective】The paper aimed to screen the suitable reference genes and to analyze and verify the expression of photosynthesis-related genes in Chenopodium album responding to Aureobasidium pullulans PA-2.【Method】The leaves of C.album that were treated for 0,1,3,5 and 7 days under PA-2 infection and the root,stem and leave of normal growth C.album were used as materials,and qRT-RCR technology and Ct value were used to assess the expression stability of three candidate reference genes(GAPDH,β-ACTIN andβ-TUBULIN).The expression levels of photosynthesis-related genes(LFRN,MDH,PetC,psaA,CAB40,psaN,BHY and MO2)were quantitatively analyzed by the optimal selected reference genes【.Result】The results indicated that GAPDH was the optimal reference gene of C.album responding to A.pullulans PA-2,and can be used for analyzing the related genes expression level to photosynthesis pathway.Using GAPDH as internal reference,the expression levels of 8 photosynthesis-related genes showed varying degrees of down-regulation,except for MDH,which reached the lowest level at 5 days.Among them,PetC,psaN,CAB40,BHY and MO2 was down-regulated C.album infected 0-1 day,reaching a high level at 3 days,and increased in the later stage.The expression levels of pasA was firstly up-regulated and then down-regulated during the infection.The expression level of LFRN was down-regulated continuously after PA-2 infection.The expression level of MDH was significantly down-regulated at first,reached the lowest level at 3 days,and increased in the later stage.In different tissues infected with PA-2,8 photosynthesis-related genes were highly expressed in leaves,among which MDH gene was highly expressed in stems,while PctC and CAB40 were the lowest expression levels in roots.Except for MDH gene,the expression levels of 7 photosynthesis-related genes in different tissues were ranked as:leaves>stems>roots.【Conclusion】The study provides foundation for the analysis of C.album genes by using qRT-RCR and the infection mechanism of A.pullulans PA-2.