本文選題：荔枝 + 衰老�。� 參考：《華南農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：荔枝(Litchi chinensis Sonn.)作為一種重要的熱帶、亞熱帶常綠果樹(shù),有著非常重要的經(jīng)濟(jì)價(jià)值。然而,在荔枝的成花過(guò)程中,由于受到外界環(huán)境及樹(shù)體自身因素的綜合影響,容易導(dǎo)致荔枝產(chǎn)量不穩(wěn)定,嚴(yán)重制約著荔枝產(chǎn)業(yè)的發(fā)展。荔枝成花質(zhì)量不佳、花序發(fā)育不良是荔枝產(chǎn)量不穩(wěn)的主要原因之一。本研究從荔枝花序上雛形葉對(duì)花序發(fā)育的影響作為切入點(diǎn),研究了‘糯米糍’與‘桂味’兩個(gè)荔枝品種在花序發(fā)育過(guò)程中,雛形葉發(fā)育與否對(duì)最終成花的影響。通過(guò)活性氧(ROS, Reactive oxygen species)處理研究‘糯米糍’花序上雛形葉衰老機(jī)理。以不同衰老階段的雛形葉進(jìn)行高通量測(cè)序,通過(guò)分析表達(dá)譜及轉(zhuǎn)錄組數(shù)據(jù),獲得了NAC及Metacaspase等與葉片衰老相關(guān)的候選基因,并利用VIGS (Virus induced gene silencing)及異源遺傳轉(zhuǎn)化技術(shù)對(duì)這些基因進(jìn)行功能研究。主要研究?jī)?nèi)容和結(jié)果如下：(1)通過(guò)生物學(xué)調(diào)查,研究荔枝‘糯米糍’和‘桂味’品種花序上雛形葉發(fā)育與否對(duì)于花序生長(zhǎng)的影響。根據(jù)不同品種花序上雛形葉對(duì)ROS及溫度響應(yīng)的差異,選擇‘糯米糍’與‘桂味’進(jìn)行成花和坐果的調(diào)查。結(jié)果表明,‘糯米糍’花序上的雛形葉對(duì)于甲基紫精(methyl viologen dichloride hydrate, MV)誘導(dǎo)的活性氧(ROS)及低溫信號(hào)敏感,在ROS脅迫及低溫誘導(dǎo)下易發(fā)生提早衰老,使得花序繼續(xù)發(fā)育,反之則雛形葉正常發(fā)育,花序萎縮甚至不發(fā)育。‘桂味’花序上雛形葉對(duì)于外源ROS及溫度信號(hào)的響應(yīng)程度弱于‘糯米糍’,花序上的雛形葉與花序可以同步發(fā)育。對(duì)2014-2015年度的田間成花情況調(diào)查顯示,雛形葉的存在嚴(yán)重影響了‘糯米糍’花序的正常發(fā)育,而對(duì)于‘桂味’則無(wú)明顯差異。利用人工溫室控溫,分別設(shè)置低溫(18/15℃)及高溫(28/25℃)環(huán)境,選擇‘糯米糍’和‘桂味’盆栽荔枝,經(jīng)過(guò)田間低溫誘導(dǎo)出現(xiàn)“白點(diǎn)”后轉(zhuǎn)入溫室生長(zhǎng)。成花情況調(diào)查表明,‘糯米糍’花序上的雛形葉在高溫環(huán)境下正常發(fā)育,低溫環(huán)境下進(jìn)入衰老狀態(tài),而‘桂味’則無(wú)顯著影響。(2)在控溫條件下,選擇經(jīng)過(guò)低溫誘導(dǎo)出現(xiàn)“白點(diǎn)”的‘糯米糍’盆栽荔枝,分別放入低溫(18/15℃)及高溫(28/25℃)溫室處理獲得不同發(fā)育方向的雛形葉和花序,對(duì)高溫下發(fā)育的雛形葉和萎縮的花序,以及低溫下發(fā)育的花序和衰老的雛形葉進(jìn)行表達(dá)譜測(cè)序分析。分析測(cè)序數(shù)據(jù)后發(fā)現(xiàn),多個(gè)差異表達(dá)基因?qū)儆贜AC類轉(zhuǎn)錄因子基因家族。對(duì)差異表達(dá)基因進(jìn)行GO分類統(tǒng)計(jì)發(fā)現(xiàn),葉片樣品之間的顯著差異基因主要富集在代謝過(guò)程(metabolic process)、細(xì)胞過(guò)程(cellular process)、蛋白代謝過(guò)程(protein metabolic proces s)等方面；而花芽樣品中,顯著差異基因主要富集在結(jié)合(binding)、細(xì)胞(cell)、代謝過(guò)程(metabolic process)等方面。(3)克隆獲得了荔枝LcNAC5.LcNAC4及LcNAP1全長(zhǎng)基因序列,NCBI比對(duì)結(jié)果表明它們均屬于NAM超級(jí)家族。通過(guò)同源比對(duì)及構(gòu)建進(jìn)化樹(shù)分析了LcNAC5, LcNAC4及LcNAP1與其他植物NAC的同源關(guān)系。同時(shí),在MV誘導(dǎo)的荔枝雛形葉衰老過(guò)程中,LcNAC5.LcNAC4及LcNAP1的相對(duì)表達(dá)量呈上升趨勢(shì)。通過(guò)遺傳轉(zhuǎn)化擬南芥及煙草,發(fā)現(xiàn)轉(zhuǎn)基因的葉片在MV誘導(dǎo)的衰老過(guò)程中,過(guò)表達(dá)LcNAC5、 LcNAC4及LcNAP1的葉片衰老速度均快于對(duì)照葉片,表明在ROS誘導(dǎo)下過(guò)表達(dá)LcNAC5,LcNAC4及LcNA1l可以加快葉片衰老進(jìn)程。(4)選取LCNAC5為研究對(duì)象,利用VIGS技術(shù)研究其在荔枝雛形葉衰老過(guò)程中的作用。通過(guò)VIGS技術(shù)沉默荔枝雛形葉中的LcNAC5,利用MV誘導(dǎo)雛形葉進(jìn)入衰老階段,發(fā)現(xiàn)沉默樣本的衰老速度遲緩于對(duì)照,且LcNAC5相對(duì)表達(dá)量顯著低于對(duì)照,表明該基因在一定程度上被沉默。同時(shí)選取了MV處理后30h的對(duì)照葉片與VIGS處理葉片進(jìn)行轉(zhuǎn)錄組測(cè)序分析,獲得了與LcNAC5沉默過(guò)程相關(guān)聯(lián)的其他差異表達(dá)基因,其中下調(diào)表達(dá)基因733個(gè),上調(diào)表達(dá)基因1857個(gè)。(5)克隆獲得了LcMCⅡ-1, RT-qPCF分析結(jié)果表明,該基因在ROS及低溫誘導(dǎo)的荔枝雛形葉衰老過(guò)程中均呈現(xiàn)出上升趨勢(shì)。對(duì)荔枝自然衰老過(guò)程的葉片進(jìn)行定量分析表明,該基因也參與了荔枝葉片自然衰老過(guò)程。利用VIGS技術(shù),在盆栽‘糯米糍’荔枝植株上進(jìn)行LcMC Ⅱ-1沉默處理,發(fā)現(xiàn)沉默處理的雛形葉對(duì)ROS誘導(dǎo)的衰老表現(xiàn)遲緩,表明LcMC Ⅱ-1參與了荔枝雛形葉衰老過(guò)程。通過(guò)遺傳轉(zhuǎn)化擬南芥,對(duì)陽(yáng)性苗的離體葉片進(jìn)行ROS處理發(fā)現(xiàn),過(guò)表達(dá)植株葉片表現(xiàn)出提早衰老的現(xiàn)象,表明ROS可以促進(jìn)過(guò)表達(dá)LcMC Ⅱ-1擬南芥葉片衰老進(jìn)程。研究結(jié)果證明了LcMC Ⅱ-1參與了荔枝雛形葉衰老過(guò)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.

【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S667.1

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 鐘揚(yáng)偉;吳淑嫻;;植物激素對(duì)糯米糍荔枝適齡結(jié)果樹(shù)促花的效應(yīng)[J];廣東農(nóng)業(yè)科學(xué);1983年05期

2 向旭，張展薇，邱燕平，袁沛元，王碧青;糯米糍荔枝坐果與內(nèi)源激素的關(guān)系[J];園藝學(xué)報(bào);1994年01期

3 田健偉;糯米糍荔枝抗逆高效栽培技術(shù)[J];柑桔與亞熱帶果樹(shù)信息;2003年02期

4 賴永超,羅詩(shī),吳美良,尹金華,羅華建;東莞糯米糍荔枝豐產(chǎn)穩(wěn)產(chǎn)綜合栽培技術(shù)[J];廣東農(nóng)業(yè)科學(xué);2002年04期

5 尹金華,羅詩(shī),賴永超,羅華建;冬季土壤含水量對(duì)糯米糍荔枝花芽分化的影響[J];亞熱帶植物科學(xué);2002年01期

6 彭堅(jiān),席嘉賓;糯米糍荔枝裂果的生理機(jī)理與防裂效果研究[J];廣西植物;2003年01期

7 尹金華;陸潔梅;黃旭明;羅詩(shī);賴永超;;糯米糍荔枝花芽誘導(dǎo)期間葉片色素和結(jié)構(gòu)的變化[J];果樹(shù)學(xué)報(bào);2008年02期

8 邱金淡;吳定堯;曾亞妮;溫丹云;武竟超;王瑜;劉偉偉;周碧燕;;土壤積水對(duì)“糯米糍”荔枝生理和果實(shí)發(fā)育的影響[J];中國(guó)南方果樹(shù);2012年04期

9 梁多;黃敏;王一凡;;火焰原子吸收光譜法測(cè)定糯米糍荔枝中的微量元素[J];農(nóng)產(chǎn)品加工(學(xué)刊);2013年02期

10 向旭,邱燕平,張展薇;糯米糍荔枝果實(shí)內(nèi)源激素與落果的關(guān)系[J];果樹(shù)科學(xué);1995年02期

相關(guān)會(huì)議論文 前2條

1 李國(guó)武;;茂名糯米糍荔枝豐產(chǎn)穩(wěn)產(chǎn)栽培技術(shù)研究[A];海峽兩岸荔枝龍眼產(chǎn)業(yè)發(fā)展研討會(huì)論文集[C];2009年

2 張哲嘉;;改進(jìn)糯米糍(‘73-S-20')荔枝生產(chǎn)之生理基礎(chǔ)與研究進(jìn)展[A];海峽兩岸荔枝龍眼產(chǎn)業(yè)發(fā)展研討會(huì)論文集[C];2009年

相關(guān)重要報(bào)紙文章 前4條

1 朱慧亮;無(wú)公害生產(chǎn)技術(shù)見(jiàn)實(shí)效 糯米糍荔枝掛果好豐碩[N];廣東科技報(bào);2005年

2 李力;從化荔枝豐收蘊(yùn)藏巨大商機(jī)[N];中華合作時(shí)報(bào);2004年

3 鄒淑玲 梁鏡財(cái);他們種的荔枝不裂果[N];廣東科技報(bào);2000年

4 本報(bào)記者 游峰　程瑋堅(jiān) 伍雪平 胡建勛 潘少婷 實(shí)習(xí)記者 胡少萍 通訊員 李耀海 付明海;有增 有減 有持平[N];東莞日?qǐng)?bào);2009年

相關(guān)博士學(xué)位論文 前1條

1 王叢叢;荔枝花序上雛形葉衰老機(jī)理的研究[D];華南農(nóng)業(yè)大學(xué);2016年

，

本文編號(hào)：1791575