荔枝花序上雏形叶衰老机理的研究

发布时间：2018-04-23 10:29

本文选题：荔枝 + 衰老　；参考：《华南农业大学》2016年博士论文

【摘要】：荔枝(Litchi chinensis Sonn.)作为一种重要的热带、亚热带常绿果树,有着非常重要的经济价值。然而,在荔枝的成花过程中,由于受到外界环境及树体自身因素的综合影响,容易导致荔枝产量不稳定,严重制约着荔枝产业的发展。荔枝成花质量不佳、花序发育不良是荔枝产量不稳的主要原因之一。本研究从荔枝花序上雏形叶对花序发育的影响作为切入点,研究了‘糯米糍’与‘桂味’两个荔枝品种在花序发育过程中,雏形叶发育与否对最终成花的影响。通过活性氧(ROS, Reactive oxygen species)处理研究‘糯米糍’花序上雏形叶衰老机理。以不同衰老阶段的雏形叶进行高通量测序,通过分析表达谱及转录组数据,获得了NAC及Metacaspase等与叶片衰老相关的候选基因,并利用VIGS (Virus induced gene silencing)及异源遗传转化技术对这些基因进行功能研究。主要研究内容和结果如下：(1)通过生物学调查,研究荔枝‘糯米糍’和‘桂味’品种花序上雏形叶发育与否对于花序生长的影响。根据不同品种花序上雏形叶对ROS及温度响应的差异,选择‘糯米糍’与‘桂味’进行成花和坐果的调查。结果表明,‘糯米糍’花序上的雏形叶对于甲基紫精(methyl viologen dichloride hydrate, MV)诱导的活性氧(ROS)及低温信号敏感,在ROS胁迫及低温诱导下易发生提早衰老,使得花序继续发育,反之则雏形叶正常发育,花序萎缩甚至不发育。‘桂味’花序上雏形叶对于外源ROS及温度信号的响应程度弱于‘糯米糍’,花序上的雏形叶与花序可以同步发育。对2014-2015年度的田间成花情况调查显示,雏形叶的存在严重影响了‘糯米糍’花序的正常发育,而对于‘桂味’则无明显差异。利用人工温室控温,分别设置低温(18/15℃)及高温(28/25℃)环境,选择‘糯米糍’和‘桂味’盆栽荔枝,经过田间低温诱导出现“白点”后转入温室生长。成花情况调查表明,‘糯米糍’花序上的雏形叶在高温环境下正常发育,低温环境下进入衰老状态,而‘桂味’则无显著影响。(2)在控温条件下,选择经过低温诱导出现“白点”的‘糯米糍’盆栽荔枝,分别放入低温(18/15℃)及高温(28/25℃)温室处理获得不同发育方向的雏形叶和花序,对高温下发育的雏形叶和萎缩的花序,以及低温下发育的花序和衰老的雏形叶进行表达谱测序分析。分析测序数据后发现,多个差异表达基因属于NAC类转录因子基因家族。对差异表达基因进行GO分类统计发现,叶片样品之间的显著差异基因主要富集在代谢过程(metabolic process)、细胞过程(cellular process)、蛋白代谢过程(protein metabolic proces s)等方面；而花芽样品中,显著差异基因主要富集在结合(binding)、细胞(cell)、代谢过程(metabolic process)等方面。(3)克隆获得了荔枝LcNAC5.LcNAC4及LcNAP1全长基因序列,NCBI比对结果表明它们均属于NAM超级家族。通过同源比对及构建进化树分析了LcNAC5, LcNAC4及LcNAP1与其他植物NAC的同源关系。同时,在MV诱导的荔枝雏形叶衰老过程中,LcNAC5.LcNAC4及LcNAP1的相对表达量呈上升趋势。通过遗传转化拟南芥及烟草,发现转基因的叶片在MV诱导的衰老过程中,过表达LcNAC5、 LcNAC4及LcNAP1的叶片衰老速度均快于对照叶片,表明在ROS诱导下过表达LcNAC5,LcNAC4及LcNA1l可以加快叶片衰老进程。(4)选取LCNAC5为研究对象,利用VIGS技术研究其在荔枝雏形叶衰老过程中的作用。通过VIGS技术沉默荔枝雏形叶中的LcNAC5,利用MV诱导雏形叶进入衰老阶段,发现沉默样本的衰老速度迟缓于对照,且LcNAC5相对表达量显著低于对照,表明该基因在一定程度上被沉默。同时选取了MV处理后30h的对照叶片与VIGS处理叶片进行转录组测序分析,获得了与LcNAC5沉默过程相关联的其他差异表达基因,其中下调表达基因733个,上调表达基因1857个。(5)克隆获得了LcMCⅡ-1, RT-qPCF分析结果表明,该基因在ROS及低温诱导的荔枝雏形叶衰老过程中均呈现出上升趋势。对荔枝自然衰老过程的叶片进行定量分析表明,该基因也参与了荔枝叶片自然衰老过程。利用VIGS技术,在盆栽‘糯米糍’荔枝植株上进行LcMC Ⅱ-1沉默处理,发现沉默处理的雏形叶对ROS诱导的衰老表现迟缓,表明LcMC Ⅱ-1参与了荔枝雏形叶衰老过程。通过遗传转化拟南芥,对阳性苗的离体叶片进行ROS处理发现,过表达植株叶片表现出提早衰老的现象,表明ROS可以促进过表达LcMC Ⅱ-1拟南芥叶片衰老进程。研究结果证明了LcMC Ⅱ-1参与了荔枝雏形叶衰老过程。
[Abstract]:Litchi chinensis Sonn., as an important tropical, subtropical evergreen fruit tree, has a very important economic value. However, in the process of litchi flower formation, due to the comprehensive influence of the external environment and the factors of the tree body itself, it is easy to lead to the instability of the litchi production. It seriously restricts the development of litchi industry. The quality of litchi flower formation. Poor inflorescence is one of the main reasons for the instability of litchi production. In this study, the effects of the rudiment leaves on the growth of the inflorescence in the inflorescence of litchi were taken as a breakthrough point. The effects of the growth and development of the two litchi varieties of 'Nuo glutinous' and' cinnamon 'in the inflorescence development were studied. The effects of the active oxygen (ROS, Reacti) on the final flower formation were studied. Ve oxygen species (species) was used to study the aging mechanism of the young leaves on the inflorescence of "glutinous glutinous". High flux sequencing was carried out at different aging stages. By analyzing the expression profiles and transcriptional data, the candidate genes related to leaf senescence were obtained, such as NAC and Metacaspase, and VIGS (Virus induced gene silencing) and heterologous genetic transformation were used. The main research contents and results are as follows: (1) through biological investigation, the effects of the development of the young leaves on the inflorescence of the "glutinous glutinous rice" and "cinnamon" varieties on the growth of the inflorescence were studied. According to the differences in the ROS and temperature responses of the young leaves on the inflorescence of different varieties, the "glutinous glutinous rice" was chosen. The results showed that the rudiment leaves on the inflorescence of the 'Nuo glutinous' were sensitive to the active oxygen (ROS) and low temperature signals induced by methyl viologen dichloride hydrate (MV). The premature aging was easy to take place under the stress of ROS stress and low temperature, and the inflorescence continued to develop, and vice versa Ye Zhengchang. The response degree of the young leaves on the exogenous ROS and the temperature signal on the inflorescence of 'cinnamon' is weaker than that of the 'glutinous rice', and the embryonic leaves and inflorescence on the inflorescence can develop synchronously. A survey of the flower formation in the field in 2014-2015 shows that the survival of the rudiment leaves seriously affected the normal hair of the inflorescence of the 'glutinous glutinous'. There was no significant difference in 'GUI Wei'. Using the artificial greenhouse temperature control, the environment was set at low temperature (18/15 C) and high temperature (28/25 C), and the "glutinous rice" and 'cinnamon' potted litchi were selected. After the "white spot" in the field, the "white spot" was turned into the greenhouse. Under the high temperature environment, the aging state was entered under the low temperature environment, while the 'GUI Wei' had no significant influence. (2) under the temperature control condition, the potted litchi of "glutinous rice glutinous", which was induced by low temperature induction, was selected to be treated at low temperature (18/15 C) and high temperature (28/25 C) to obtain the embryonic leaves and inflorescence of different developmental directions. The expression profiles of the embryonic and atrophic inflorescence developed at high temperature, as well as the inflorescence and the embryonic leaves developed at low temperature were sequenced and analyzed. After the analysis of the sequencing data, a number of differentially expressed genes were found to belong to the NAC family of transcription factor genes. The significant differences among the leaf samples were found by the GO classification of differentially expressed genes. The genes are mainly enriched in the metabolic process (metabolic process), the cell process (cellular process), the protein metabolism process (protein metabolic proces s) and so on. In the flower bud samples, the significant difference genes are mainly enriched in the combination (binding), cell (cell), and the metabolic process (metabolic process). (3) the clones obtained the litchi C4 and LcNAP1 full-length gene sequences, NCBI alignment results showed that both of them belong to the NAM superfamily. The homologous relationship between LcNAC5, LcNAC4 and LcNAP1 with other plant NAC was analyzed by homologous alignment and construction of the evolutionary tree. Meanwhile, the relative expression of LcNAC5.LcNAC4 and LcNAP1 increased in the process of MV induced early leaf senescence of litchi. Genetic transformation of Arabidopsis and tobacco showed that the leaf senescence of LcNAC5, LcNAC4 and LcNAP1 was faster than that of control leaves during the aging process of MV induced by transgenic leaves. It showed that under ROS induced LcNAC5, LcNAC4 and LcNA1l could accelerate the process of leaf senescence. (4) LCNAC5 was selected as the research object, and VIGS technology was used to study it. In the process of aging of litchi young leaves, the VIGS technique was used to silence the LcNAC5 in the young litchi leaves, and MV was used to induce the embryonic leaves to enter the aging stage. The aging speed of the silent samples was delayed to the control, and the relative expression of LcNAC5 was significantly lower than that of the control, indicating that the gene was silenced to a certain extent. At the same time, the 30h was selected to be treated with the MV treatment. The control leaves and the VIGS treated leaves were sequenced and analyzed. Other differentially expressed genes associated with the LcNAC5 silencing process were obtained, including 733 down-regulated genes and 1857 up-regulated genes. (5) the clone obtained LcMC II -1. The RT-qPCF analysis showed that the gene was senescent at ROS and low temperature induced litchi shaped leaves. Cheng Zhongjun showed an upward trend. Quantitative analysis of the leaves of natural senescence of litchi showed that the gene was also involved in the natural aging process of litchi leaves. Using VIGS technology, LcMC II -1 was used in the potted 'glutinous' Litchi plants to be treated with LcMC II -1, and it was found that the embryonic leaves of the silent treatment were slow to the senescence induced by ROS. LcMC II -1 participated in the aging process of the young litchi leaves. Through genetic transformation of Arabidopsis, the ROS treatment of the leaves of the positive seedlings showed that the overexpressed plant leaves showed early senescence, indicating that ROS could promote the over expression of the senescence of LcMC II -1 Arabidopsis leaves. The results showed that LcMC II -1 participated in the young litchi young leaves. The process of aging.

【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S667.1

【相似文献】