大豆胞囊线虫侵染下野生大豆根组织实时定量PCR分析中内参基因的筛选

Reference Gene Identification for Real-time Quantitative PCR Analysis in Glycine soja Roots Infected with Heterodera glycine

以高抗和高感HG 0大豆胞囊线虫的野生大豆种质为试验材料,采用q RT-PCR技术检测了ACT11(Glyma.18G290800)、Tubulin-motif(Glyma.19G194800)等24个候选持家基因在不同大豆胞囊线虫侵染时期(接种后9,15和20 d)野生大豆根系中的基因表达情况,除Actin(Glyma.08G182200)出现了非特异扩增外,其它23个持家基因的q RT-PCR扩增效果均理想,扩增特异性高。分析上述23个持家基因的表达丰度,并利用Best Keeper、ge Norm和Normfinder对其表达稳定性进行评价。结果表明:持家基因间的表达丰度不尽相同,而且有些持家基因在不同品种间或不同处理间(接种虫卵和接种水)也存在表达丰度的差异。23个持家基因的表达稳定性也不相同,综合来看,表达最不稳定的基因为Cons9(Glyma.10G152200)、Cons2(Glyma.17G138500)、Cons1(Glyma.15G270900)和Tubulin-motif(Glyma.20G136000),本试验条件下它们不适宜作内参基因;其余持家基因相对稳定,本试验条件下可选择Tubulinmotif(Glyma.15G132200),Cons6/SKIP16(Glyma.12G051100)或Tubulin-motif(Glyma.05G110200)作为内参基因,它们表达量较高,表达最为稳定,其Ct平均值分别为25.7、26.5和25.0,标准偏差分别为0.540、0.575和0.490,ge Norm软件评价其稳定性的M值分别为0.698、0.715和0.727。该研究为野生大豆抗胞囊线虫相关基因的表达分析提供了参考。

Real-time quantitative PCR （qRT-PCR） has been widely used in crops for gene expression analysis, and reference genes are required in qRT-PCR analysis to minimize influences of RNA quality and quantity and efficiency of reverse transcription. Housekeeping genes are often selected as reference genes, however many of the housekeeping genes provide stable expression under only certain environments. So it is necessary to identify and select suitable reference genes for a given experiment. In our study, 24 candidate housekeeping genes were selected for their gene expression analysis, there happened nonspe- cific amplification products for qRT-PCR of Actin（ Glyma. 08G182200）. The other 23 candidate housekeeping genes were evaluated for their expression level and stability in roots of susceptible and resistant wild soybean accessions under soybean cyst nematode HG 0 infection at 9, 15 and 20 days after inoculation. Housekeeping genes had different expression level, and some of them had different expression level between wild soybean accessions, or between different treatments. There also existed difference in expression stability among the 23 housekeeping genes. As a whole, Cons9 （ Glyma. 10G152200 ）, Cons2 （ Glyma. 17G138500）, Cons1（ Glyma. 15G270900） and Tubulin_motif （ Glyma. 20G136000） were the top four least stable genes, and they were not suitable for the study. The three housekeeping genes of Tubulinmotif （ Glyma. 15G132200） , Cons6/SKIP16 （Glyma. 12G051100） and Tubulin_motif （Glyma. 05Gl10200） were the most stable genes with M value of 0. 698, 0. 715 and 0. 727 respectively and with their expression level of Ct = 25.7, 26. 5 and 25.0 respectively, which indicated that they are ideal reference genes for qRT-PCR analysis under our experiment conditions.