芒果细菌性角斑病菌在侵染芒果叶片过程中内参基因筛选  

Screening of Reference Genes in Xanthomonas citri pv.mangiferaeindicae During the Infection of Mango Leaf

作　　者：姚全胜[1] 杨倩 柳凤[1] 詹儒林[1] Yao Quansheng;Yang Qian;Liu Feng;Zhan Rulin(Key Laboratory of Tropical Fruit Biology,Ministry of Agriculture,Key Laboratory for Post-harvest Physiology and Technology of Tropical Horticultural Products of Hainan Province,South Asia Tropical Crop Research Institute,CATAS,Zhanjiang,524091)

机构地区：[1]中国热带农业科学院南亚热带作物研究所,海南省热带园艺产品采后生理与保鲜重点实验室,农业部热带果树生物学重点实验室,湛江524091

出　　处：《分子植物育种》2021年第18期6088-6095,共8页Molecular Plant Breeding

基　　金：国家重点研发计划(2019YFD1001100);中央非盈利性研究机构基本科研业务费专项(1630062020014)共同资助。

摘　　要：为筛选芒果细菌性角斑病菌在侵染芒果叶片过程中稳定表达的内参基因,采用实时荧光定量聚合酶链式扩增反应技术检测和分析病原细菌的16S rRNA、gyrB、GAPDH、dan K和rpo D这5个看家基因的表达量。结果表明,经过PCR扩增效率筛选,5个基因均扩增出单一条带,且条带大小与预期产物相符,特异性较好,符合基本稳定性要求。利用ge Norm软件分析发现,GAPDH和rpo D稳定性最高,M值均为0.122。其余3个基因的表达稳定性依次为gyr B(M=0.17)>dan K(M=0.325)>16S rRNA(M=0.542)。在5个候选内参基因中,GAPDH和gyr B为病原细菌侵染芒果叶片下表达最稳定的基因,且不需要引入第三个基因(V2/3=0.063)。NormFinder软件分析5个候选内参基因稳定性数值顺序依次为:gyr B(0.092)>dan K(0.169)>GAPDH(0.188)>rpo D(0.235)>16S rRNA(0.581)。Bestkeeper程序分析5个候选内参基因的标准差(SD)值都在0.86~0.79之间。综合以上结果发现GAPDH和gyr B基因的表达稳定性最好,能够作为病原细菌在侵染芒果叶片时的内参基因,更准确地校正定量结果。In order to screen out the best reference genes for(real-time quantitative polymerase chain reaction,RT-qPCR)normalization in Xanthomonas citri pv.mangiferaeindicae under infection mango leaf.The m RNA differential expression of 5 traditional reference genes,including 16 S rRNA,gyr B,GAPDH,dan K and rpo D of X.citri pv.mangiferaeindicae were systematically compared by RT-qPCR.According to PCR amplification efficiencies,5 housekeeping genes were expression stabilities were evaluated by computer algorithms geNorm,NormFinder and BestKeeper program.The results showed that,after PCR amplification efficiency screening,a single band was amplified from all the 5 genes,and the band size was consistent with the expected product,with good specificity and meeting the basic stability requirements.As determined by geNorm,GAPDH and rpo D were the most stable reference genes,with average expression stability value of 0.122.The expression stability of the remaining 3 genes was gyr B(M=0.17)>dan K(M=0.325)>16 S rRNA(M=0.542).GAPDH and gyr B were the most stable genes of mango leaf under X.citri pv.mangiferaeindicae infection while the third gene was not need to introduce.BestKeeper revealed that gyr B was the most stables references gene.The stability of reference gene were ranked as gyr B(0.092)>dan K(0.169)>GAPDH(0.188)>rpo D(0.235)>16 S rRNA(0.581).Analysis 5 candidate reference genes by BestKeeper,the SD values are between 0.86 and 0.79,all genes were stable.Three software analysis revealed that the most stable genes were GAPDH and gyr B.they showed their housekeeping genes features’and could be used jointly as reference genes of X.citri pv.mangiferaeindicae for plant-microb interaction analysis.

关 键 词：芒果细菌性角斑病菌 侵染 实时荧光定量聚合酶链式扩增反应 内参基因 

分 类 号：S436.67[农业科学—农业昆虫与害虫防治]