玉米ZmPP6C基因的克隆及其响应光和胁迫处理的表达分析

发布时间：2018-07-04 07:31

本文选题：玉米 + 丝氨酸/苏氨酸蛋白磷酸酶　；参考：《河南农业大学》2016年硕士论文

【摘要】：丝氨酸/苏氨酸蛋白磷酸酶6亚基(catalytic subunits of Ser/Thr protein phosphatase 6,PP6C)是PP6全酶的催化亚基。在模式植物拟南芥中的研究表明,PP6C参与生长素极性运输、脱落酸信号转导和光信号转导途径介导的开花调控。本研究采用RT-PCR方法克隆了ZmPP6C基因,分析了蛋白结构特征与同源蛋白间的进化关系,利用定量PCR的方法分析了ZmPP6C基因对不同光质、黑暗到各种光质转换、长日照和短日照处理,以及胁迫处理下的表达模式,结果如下:1.蛋白磷酸酶6亚基的氨基酸序列在进化上高度保守。序列分析表明,ZmPP6C基因开放阅读框为912个核苷酸,编码303个氨基酸残基,包含PP2A的催化亚基PP2Ac结构域;系统进化树分析表明,PP6C蛋白在进化上较为保守,无论是单子叶植物还是双子叶植物,氨基酸序列高度相似,可达91%。并且与高粱的PP6C蛋白相似性更高,高达99.67%。2.ZmPP6C基因在玉米叶片中表达量较高,并且响应不同光质处理。对玉米自交系B73的ZmPP6C基因进行器官特异性表达分析表明,其表达量在成株期叶片中最高,是根中的7.9倍,在茎、雄花、叶枕、叶鞘和花柄中的表达量相对较低;ZmPP6C能够响应不同光质处理,且受远红光和红光的影响较大。ZmPP6C的表达量在黑暗转入远红光30分钟达到峰值,然后迅速下降,直到24 h一直保持较低的水平;转入红光15分钟达到峰值,迅速下降后缓慢上升,直到24 h达到黑暗的7.2倍。3.ZmPP6C基因转录丰度能响应长日照和短日照处理,暗示其可能通过光信号转导通路来调控植物开花。在长日照条件下,玉米ZmPP6C的表达在长日条件下的光照和黑暗阶段各有一个明显的表达高峰,分别出现在进入光照阶段后10 h和进入黑暗阶段5 h时。在短日照条件下,ZmPP6C基因的表达丰度波动起伏较大,最高峰值发生在进入黑暗后10 h,次高峰值发生在进入光照后8 h时。4.ZmPP6C基因转录丰度受胁迫处理的调节。在高渗透、盐渍和淹水胁迫过程中,ZmPP6C基因的表达水平先降后升。B73幼苗在200 m M NaCl胁迫处理12 h、24 h和48 h时,ZmPP6C的转录丰度是未处理的0.8倍、1.1倍和2.6倍。B73幼苗在20%PEG6000处理20 h时,ZmPP6C基因的表达水平与未处理的相当;48 h时,ZmPP6C的表达水平上升到未处理的的4.5倍。淹水处理处理5 d和7 d,ZmPP6C的表达分别上升到未处理的1.3倍和4.1倍;然后恢复正常温室生长条件3 d和7 d,ZmPP6C的表达上升到未处理的3.0倍和6.9倍。表明ZmPP6C基因参与了玉米胁迫的应答。
[Abstract]:Serine / threonine protein phosphatase 6 (catalytic subunits of Serr protein phosphatase 6 PP6C) is the catalytic subunit of PP6 whole enzyme. Studies in Arabidopsis thaliana showed that PP6C was involved in polar transport of auxin, abscisic acid signal transduction and light signal transduction mediated flowering regulation. In this study, the ZmPP6C gene was cloned by RT-PCR, and the evolutionary relationship between the protein structure and the homologous proteins was analyzed. The effects of different light quality, dark to various light quality, long sunlight and short sunlight were analyzed by quantitative PCR. And the expression pattern under stress, the result is as follows: 1. The amino acid sequence of protein phosphatase 6 subunit is highly conserved in evolution. Sequence analysis showed that the open reading frame of ZmPP6C gene was 912 nucleotides, encoding 303 amino acid residues, containing the catalytic subunit PP2Ac domain of PP2A, and phylogenetic tree analysis showed that PPP6C protein was more conserved in evolution. The amino acid sequences of monocotyledonous and dicotyledonous plants are similar, up to 91. The PP6C protein of sorghum was more similar to that of sorghum, as high as 99.67.2.ZmPP6C gene was expressed in maize leaves and responded to different light quality treatments. The organ-specific expression of ZmPP6C gene in maize inbred line B73 was analyzed. The results showed that the expression of ZmPP6C gene was the highest in adult leaves, 7.9 times higher than that in root, and 7.9 times in stem, male flower and leaf pillow. The expression of ZmPP6C in leaf sheath and petiole was relatively low, and the expression of ZmPP6C was significantly affected by far red light and red light. The expression of ZmPP6C reached its peak value at 30 minutes after the dark light was transferred to the far red light, and then decreased rapidly. The transcriptional abundance of ZmPP6C gene reached a peak level at 24 h, reached a peak value at 15 minutes of red light, decreased rapidly and increased slowly, until 24 h after transfer to dark 7.2-fold. 3. ZmPP6C gene transcription abundance could respond to long sunlight and short day exposure. It suggests that it may regulate plant flowering through the light signal transduction pathway. Under the condition of long sunlight, the expression of ZmPP6C in maize had an obvious peak in the light and dark stage, which appeared at 10 h after the light stage and 5 h after the dark stage, respectively. The expression abundance of ZmPP6C gene fluctuated greatly under the condition of short sunlight, the highest peak occurred at 10 hours after darkness, and the second peak occurred at 8 h after exposure to light. 4. The transcription abundance of ZmPP6C gene was regulated by stress treatment. At high penetration, The expression level of ZmPP6C gene decreased at first and then increased under 200 mm NaCl stress. The transcription abundance of ZmPP6C was 1.1 times higher than that of untreated ZmPP6C at 24 h and 48 h after 200mm NaCl stress, and 2.6-fold. B73 was ZmPP6C when treated with 20b PEG6000 for 20 h. The transcriptional abundance of ZmPP6C was 1.1 times and 2.6 times higher than that of untreated seedlings at 20 min PEG6000 for 20 h. The expression level of ZmPP6C was 4.5 times higher than that of untreated ZmPP6C at 48 h. The expression of ZmPP6C increased to 1.3 times and 4.1 times of untreated after 5 days and 7 days of flooding respectively, and then increased to 3.0 and 6.9 times of untreated after 3 days and 7 days of normal greenhouse growth conditions. The results showed that ZmPP6C gene was involved in maize stress response.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S513;Q943.2

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