Selection Of Candidate Reference Genes For Gene Expression Studies By RT-qPCR In Soybean

Soybean (Glycine max (L.) Merr) is an important alimentarn crop, it has more than 5000 years of cultural history. Researches of soybean starts early. Recent years, along with the development of biotechnology, the main trend of soybean scientific research is focusing on molecular biology. The study of soybean physiological and biochemical processes, which plays an important role in soybean germplasm improvement, is studying by gene expression. Real-time quantitative PCR (RT-qPCR), as an accurate test methods for nucleic acid quantification, is widly used in the study of gene expression. Reference gene (RG) plays an important role in standardization of transcript abundance, besides, it is extremely effect the accuracy of RT-qPCR result. This study intend to use five traditional RGs:ACT11, GAPDH, TUB4, EF1A, CYP2 and five new RGs from different test method:MTP, MTP1, CDPKK, ABCT, UKN2 as candidate RGs. (Part of them from published study, part own design). The result of primer test showed that MTP was mismatched, ABCT had a double melting peaks. Than, the left eight primers was screened out for the next experiment. RT-qPCR experiments were carry out with following materials:Development and germination stage of soybean seed materials, consists of seed, cotyledon and axes. Vegetative materials:include root, stem and leaf. Stress conditions:drought stress, high temperature stress. Results were comprehensive analysis by geNorm, NormFinder and Bestkeeper. Different environments, different tissues optimum reference gene or combination of genes were selected. The results are as follows:1) In seed development and germination stage, CYP2, GAPDH, CDPKK is the most stable. Expression RG; and in seed CYP2 GAPDH is the proper RG; in cotyledons ACT11 and CYP2 has best expression stability; in axes recommended UKN2 and TUB4 as RGs.2) A comprehensive analysis of vegetative organs recommended EF1A, CYP2 and ACT11 in combination; recommended ACT11, CDPKK and TUB4 as the best RGs of root; and in the stem TUB4 CYP2 is the optimum reference gene; in the leaves the best reference gene is CYP2 and UKN2.3) Under stress condition, UKN2, TUB4 and EF1A is considered as proper RGs. Drought stress condition recommended EF1A, CYP2 and TUB4 used in conjunction. TUB4, UKN2 and MTPlhad best performance under high temperatures condition.4) All the sample pools result seems that there is no way to accurately filter out one or a set of reference gene which is constant expressed.Combined with previous studies, may be used in CYP2 and ACTll can make the RT-qPCR results of all materials more accuracy.