Zhong-Wei Zhang1, Ling-Yang Feng2, Jian-Hui Wang3, Yu-Fan Fu1, Xin Cai1, Chang-Quan Wang1, Jun-Bo Du2, Ming Yuan4, Yang-Er Chen4, Pei-Zhou Xu5, Ting Lan1, Guang-Deng Chen1, Lin-Tao Wu6, Yun Li7, Jin-Yao Hu8, Shu Yuan9. 1. College of Resources, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. 2. College of Agronomy, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. 3. Horticulture Institute, Sichuan Academy of Agricultural Sciences, Chengdu, 610066, Sichuan, China. 4. College of Life Sciences, Sichuan Agricultural University, Ya'an, 625014, Sichuan, China. 5. Rice Research Institute, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. 6. Rape Research Institute, Guizhou Academy of Agricultural Sciences, Guiyang, 550008, Guizhou, China. 7. Rape Research Institute, Chengdu Academy of Agriculture and Forestry, Chengdu, 611130, Sichuan, China. 8. Research Center for Eco-Environmental Engineering, Mianyang Normal University, Mianyang, 621000, Sichuan, China. jinyaohu@126.com. 9. College of Resources, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. roundtree318@hotmail.com.
Abstract
MAIN CONCLUSION: The 5-leaf-stage rape seedlings were more insensitive to Pi starvation than that of the 3-leaf-stage plants, which may be attributed to the higher expression levels of ethylene signaling and sugar-metabolism genes in more mature seedlings. Traditional suppression subtractive hybridization (SSH) and RNA-Seq usually screen out thousands of differentially expressed genes. However, identification of the most important regulators has not been performed to date. Here, we employed two methods, namely, a two-round SSH and two-factor transcriptome analysis derived from the two-factor ANOVA that is commonly used in the statistics, to identify development-associated inorganic phosphate (Pi) starvation-induced genes in Brassica napus. Several of these genes are related to ethylene signaling (such as EIN3, ACO3, ACS8, ERF1A, and ERF2) or sugar metabolism (such as ACC2, GH3, LHCB1.4, XTH4, and SUS2). Although sucrose and ethylene may counteract each other at the biosynthetic level, they may also work synergistically on Pi-starvation-induced gene expression (such as PT1, PT2, RNS1, ACP5, AT4, and IPS1) and root acid phosphatase activation. Furthermore, three new transcription factors that are responsive to Pi starvation were identified: the zinc-finger MYND domain-containing protein 15 (MYND), a Magonashi family protein (MAGO), and a B-box zinc-finger family salt-tolerance protein. This study indicates that the two methods are highly efficient for functional gene screening in non-model organisms.
MAIN CONCLUSION: The 5-leaf-stage rape seedlings were more insensitive to Pi starvation than that of the 3-leaf-stage plants, which may be attributed to the higher expression levels of ethylene signaling and sugar-metabolism genes in more mature seedlings. Traditional suppression subtractive hybridization (SSH) and RNA-Seq usually screen out thousands of differentially expressed genes. However, identification of the most important regulators has not been performed to date. Here, we employed two methods, namely, a two-round SSH and two-factor transcriptome analysis derived from the two-factor ANOVA that is commonly used in the statistics, to identify development-associated inorganic phosphate (Pi) starvation-induced genes in Brassica napus. Several of these genes are related to ethylene signaling (such as EIN3, ACO3, ACS8, ERF1A, and ERF2) or sugar metabolism (such as ACC2, GH3, LHCB1.4, XTH4, and SUS2). Although sucrose and ethylene may counteract each other at the biosynthetic level, they may also work synergistically on Pi-starvation-induced gene expression (such as PT1, PT2, RNS1, ACP5, AT4, and IPS1) and root acid phosphatase activation. Furthermore, three new transcription factors that are responsive to Pi starvation were identified: the zinc-finger MYND domain-containing protein 15 (MYND), a Magonashi family protein (MAGO), and a B-box zinc-finger family salt-tolerance protein. This study indicates that the two methods are highly efficient for functional gene screening in non-model organisms.
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