Abstract
Annual ryegrass (Lolium multiflorum) is a cool-season annual grass cultivated worldwide for its high yield and quality. With the areas of saline soil increasing, investigation of the molecular mechanisms of annual ryegrass tolerance under salt stress has become a significant topic. qRT-PCR has been a predominant assay for determination of the gene expression, in which selecting a valid internal reference gene is a crucial step. The objective of present study was to evaluate and identify suitable reference genes for qRT-PCR in annual ryegrass under salt stress. The results calculated by RefFinder indicated that eEF1A(s) was the most stable reference gene in leaves, whereas EF1-a was the least stable; meanwhile, TBP-1 was the most optimal in roots and in all samples, and the eIF-5A shouldn’t be utilized for normalization of the gene expression. eEF1A(s) is more suitable than TBP-1 as reference gene in leaves when verified with P5CS1 and Cyt-Cu/Zn SOD genes. We should choose optimal reference genes in specific tissues instead of the most stable one selected from different conditions and tissues.
Highlights
Quantitative reverse transcription PCR or quantitative real-time RT-PCR is a well-developed and popular molecular biology technique to rapidly and precisely detect gene expression by measuring relative mRNA levels in cells [1,2,3]
Numerous reference genes are used as internal control genes for the Poaceae, such as actin [4], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [5,6], YT521-B-like protein family protein (YT521-B), eukaryotic elongation factor 1 alpha (eEF1A(s)) [7], and TATA binding protein (TBP-1) [8]
For perennial ryegrass, eEF1A (s) and YT521-B were regarded as suitable reference genes with different defoliation management [11]; eIF-4a and TEF1 were considered the most stably expressed genes in drought stress and ABA treatment conditions [12], so eIF4A and TBP-1 should be used as housekeeping genes (HKGs) under salt and heat stress [12]
Summary
Quantitative reverse transcription PCR or quantitative real-time RT-PCR (qRT-PCR) is a well-developed and popular molecular biology technique to rapidly and precisely detect gene expression by measuring relative mRNA levels in cells [1,2,3]. Numerous reference genes are used as internal control genes for the Poaceae, such as actin [4], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [5,6], YT521-B-like protein family protein (YT521-B), eukaryotic elongation factor 1 alpha (eEF1A(s)) [7], and TATA binding protein (TBP-1) [8] These HKGs or references genes should be expressed at the same level in different tissues, at all developmental stages or in different experiments. Researchers prefer to utilize the traditional method—clone and verify reference genes through the sequence in relative and model plants We used this method to select valid reference genes depending on different tissues, cells and experimental treatments for accurate and optimal results [15,16,17,18]. Genes data, which will be beneficial for taking effective measures to change or to rationally use the saline soil in time
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
