用于立枯丝核菌感染及生物防治促生菌/硅酸钾施用的水稻RT-qPCR分析的内参基因选择

Selection of reference genes for RT-qPCR analysis of rice with Rhizoctonia solani infection and biocontrol PGPR/KSi application.

作者信息

Hashemipetroudi Seyyed Hamidreza, Ghorbani Hamidreza, Rostami Mehdi, Rezaei Ali, Goodwin Paul H, Kuhlmann Markus

机构信息

Department of Genetics Engineering and Biology, Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari Agricultural Sciences and Natural Resources University, Sari, Iran.

Crop and Horticultural Science Research Department, Mazandaran Agricultural and Natural Resources Research and Education Center, AREEO, Sari, Iran.

出版信息

Mol Biol Rep. 2023 May;50(5):4225-4237. doi: 10.1007/s11033-023-08361-0. Epub 2023 Mar 10.

DOI:10.1007/s11033-023-08361-0

PMID:36894770

Abstract

BACKGROUND

Rhizoctonia solani (AG1 IA) is an important pathogen of rice (Oryza sativa L.) that causes rice sheath blight (RSB). Since control of RSB by breeding and fungicides have had limited success, novel strategies like biocontrol with plant growth-promoting rhizobacteria (PGPR) can be an effective alternative.

METHOD AND RESULTS

Seven commonly used reference genes (RGs), 18SrRNA, ACT1, GAPDH2, UBC5, RPS27, eIF4a and CYP28, were evaluated for their stability in rice-R. solani-PGPR interaction for real-time quantitative PCR (RT-qPCR) analysis. Different algorithms were examined, Delta Ct, geNorm, NormFinder, BestKeeper, and comprehensive ranking by RefFinder, to evaluate RT-qPCR of rice in tissues infected with R. solani and treated with the PGPR strains, Pseudomonas saponiphilia and Pseudomonas protegens, with potassium silicate (KSi) alone or in combination with each PGPR strain. RG stability was affected for each treatment and treatment-specific RG selection was suggested. Validation analysis was done for nonexpressor of PR-1(NPR1) for each treatment.

CONCLUSION

Overall, ACT1 was the most stable RG with R. solani infection alone, GAPDH2 with R. solani infection plus KSi, UBC5 with R. solani infection plus P. saponiphilia, and eIF4a with R. solani infection plus P. protegens. Both ACT1 and RPS27 were the most stable with the combination of KSi and P. saponiphilia, while RPS27 was the most stable with the combination of KSi and P. protegens.

摘要

背景

立枯丝核菌（AG1 IA）是水稻（Oryza sativa L.）的一种重要病原菌，可引发水稻纹枯病（RSB）。由于通过育种和杀菌剂防治水稻纹枯病的成效有限，利用促植物生长根际细菌（PGPR）进行生物防治等新策略可能是一种有效的替代方法。

方法与结果

评估了7个常用内参基因（RGs），即18SrRNA、ACT1、GAPDH2、UBC5、RPS27、eIF4a和CYP28在水稻-立枯丝核菌-PGPR互作中的稳定性，用于实时定量PCR（RT-qPCR）分析。研究了不同算法，即Delta Ct、geNorm、NormFinder、BestKeeper以及通过RefFinder进行综合排名，以评估在感染立枯丝核菌并用PGPR菌株嗜皂假单胞菌和荧光假单胞菌处理的水稻组织中，单独使用硅酸钾（KSi）或与每种PGPR菌株组合使用时的RT-qPCR情况。每种处理都会影响内参基因的稳定性，并建议针对特定处理选择合适的内参基因。对每种处理的PR-1非表达子（NPR1）进行了验证分析。

结论

总体而言，单独感染立枯丝核菌时ACT1是最稳定的内参基因；感染立枯丝核菌加KSi时GAPDH2最稳定；感染立枯丝核菌加嗜皂假单胞菌时UBC5最稳定；感染立枯丝核菌加荧光假单胞菌时eIF4a最稳定。KSi与嗜皂假单胞菌组合时ACT1和RPS27都是最稳定的，而KSi与荧光假单胞菌组合时RPS27是最稳定的。