Abstract
Physic nut (Jatropha curcas L) seed oil is a natural resource for the alternative production of fossil fuel. Seed oil production is mainly depended on seed yield, which was restricted by the low ratio of staminate flowers to pistillate flowers. Further, the mechanism of physic nut flower sex differentiation has not been fully understood yet. Quantitative Real Time—Polymerase Chain Reaction is a reliable and widely used technique to quantify the gene expression pattern in biological samples. However, for accuracy of qRT-PCR, appropriate reference gene is highly desirable to quantify the target gene level. Hence, the present study was aimed to identify the stable reference genes in staminate and pistillate flowers of J. curcas. In this study, 10 candidate reference genes were selected and evaluated for their expression stability in staminate and pistillate flowers, and their stability was validated by five different algorithms (ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder). Resulting, TUB and EF found to be the two most stably expressed reference for staminate flower; while GAPDH1 and EF found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene AGAMOUS using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. AGAMOUS gene expression levels at different stages were further proved by gene copy number analysis. Therefore, the present study provides guidance for selecting appropriate reference genes for analyzing the expression pattern of floral developmental genes in staminate and pistillate flowers of J. curcas.
Highlights
Physic nut (Jatropha curcas L) is a multipurpose, poisonous, drought resistant, semi-evergreen shrub/small tree belonging to the family Euphorbiaceae and it has spread beyond its center of origin to tropical and subtropical regions because of its simple propagation, rapid growth, PLOS ONE | DOI:10.1371/journal.pone.0172460 February 24, 2017Identification and validation of superior reference gene in Jatropha curcas flowers
The flowers were collected from physic nut plants grown at Jinhe town (26 ̊560N, 101 ̊680E) located in Yanyuan County, Sichuan Province, China (Permission provided by Yanyuan county Government, China) and stem cutting grown at Green house, College of Life Sciences, Sichuan University, China, collected from Haikou city, Hainan Province (Permission provided by Haikou city Government, China) (S1 Fig)
Useful endogenous reference genes were selected based on previous studies, which are generally used for normalization and routinely used as control for RT-PCR or blotting techniques
Summary
The flowers were collected from physic nut plants grown at Jinhe town (26 ̊560N, 101 ̊680E) located in Yanyuan County, Sichuan Province, China (Permission provided by Yanyuan county Government, China) and stem cutting grown at Green house, College of Life Sciences, Sichuan University, China, collected from Haikou city, Hainan Province (Permission provided by Haikou city Government, China) (S1 Fig). One inflorescence was collected from one J. curcas plant and totally 20 inflorescence samples were randomly collected each time from J. curcas. Two to three staminate and pistillate flowers were collected from one inflorescence separately in earlier, middle and later according to the flower developmental stages described by Wu et al [15] (S2 Fig). The flowers of earlier stages were separated from inflorescence under the stereo microscope (Olympus, SZ2). The collected flower samples were immediately frozen in liquid nitrogen and stored at −80 ̊C for further experimental analysis
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