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. 2016 Jul 29:7:1149.
doi: 10.3389/fpls.2016.01149. eCollection 2016.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.)

Jinquan Chao  1 , Shuguang Yang  1 , Yueyi Chen  1 , Wei-Min Tian  1
Affiliations

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.)

Jinquan Chao et al. Front Plant Sci. .

Abstract

Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR.

Keywords: Hevea brasiliensis Muell. Arg.; NormFinder software; geNorm software; latex flow; qRT-PCR; reference gene.

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Figures

FIGURE 1
FIGURE 1
Latex flow of rubber tree (A) and the difference in latex characters (B,C) between CATAS7-33-97 and CATAS8-79. (A) Latex flow of rubber tree after tapping. (B) The difference in duration of latex flow between CATAS7-33-97 and CATAS8-79. (C) The difference in latex production between CATAS7-33-97 and CATAS8-79. Significant difference was indicated by the asterisks above the bars (∗∗∗p < 0.001).
FIGURE 2
FIGURE 2
Average cycle threshold (Ct) values for the 23 candidate reference genes upon duration of latex flow.
FIGURE 3
FIGURE 3
Average gene expression stability (M) and pairwise variation (V) analyses of the candidate reference genes in different sample sets generated by the geNorm software. The ranking of the gene expression stability performed in CATAS7-33-97 (A), CATAS8-79 (B) and total sample (C). The most stable genes are on the right, and the least stable genes are on the left. (D) Pairwise variation calculated to determine the minimum number of reference genes for accurate normalization.
FIGURE 4
FIGURE 4
Relative expression of HbHMGR1(A), HbSRPP(B), HbRpb11(C) and HbTFIIB(D) by using 18S rRNA, FP or UBC2b for normalization. Error bars for qRT-PCR showed the standard deviation of three replicates. Different letters indicate statistically significant difference in relative expression of each gene among different tissues. The capital letter represents p < 0.01 while lowercase represents p < 0.05. Analyses were performed with SPSS Statistics 17.0. Bars indicate the standard deviation of three biological replicates in triplicate.
FIGURE 5
FIGURE 5
Comparing suitable reference genes screened here with previous studies on rubber tree. The top five best references of each experimental group were analyzed here.
See this image and copyright information in PMC

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