本文選題：天藍(lán)苜蓿 + 銅脅迫　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：重金屬脅迫對共生固氮的影響,目前多停留在表型觀察及生理數(shù)據(jù)分析,對其分子機(jī)制方面的研究鮮有報道。本研究以豆科植物天藍(lán)苜蓿作為研究對象,對實驗室前期通過RNA-seq分離到的銅脅迫下天藍(lán)苜蓿共生根中差異表達(dá)的部分基因,分別進(jìn)行銅脅迫及共生過程的表達(dá)分析,檢測它們與銅脅迫及共生的關(guān)系;同時,利用RNA干擾技術(shù),對前期驗證過的一個與銅脅迫相關(guān)且在接菌根中特異表達(dá)的幾丁質(zhì)酶編碼基因Cu33611,進(jìn)行結(jié)瘤過程的功能鑒定。研究內(nèi)容及得到的結(jié)果簡述如下:1.編碼富含半胱氨酸簇的蛋白質(zhì)(cysteine cluster protein,CCP)基因被認(rèn)為與防御和共生相關(guān)。選擇9個CCP基因進(jìn)行共生過程的表達(dá)分析,9個CCP基因的表達(dá)大體上可分為三類:ML68936_c0和ML94029_c0的表達(dá)水平在接菌后7d達(dá)到或接近最高水平,隨后下降,直至接菌后30d恢復(fù)高水平表達(dá),主要在結(jié)瘤的早期和晚期表達(dá);ML67778_c0、ML85596_c0、ML98221_c0、ML101635_c0和ML68801_c0在根瘤形成中期表達(dá)量較高,接菌后30d時達(dá)到最高,可能在結(jié)瘤中期和晚期起作用;ML54082_c0、ML64613_c0和ML90197_c0則在根瘤形成晚期表達(dá)量較高,其他檢測時間點不表達(dá)或低水平表達(dá),推測其主要在根瘤形成晚期發(fā)揮作用。2.與乙烯合成及信號轉(zhuǎn)導(dǎo)相關(guān)基因的表達(dá)分析顯示,乙烯合成有關(guān)的基因ML98991_c3(氨基環(huán)丙烷羧酸氧化酶,ACO)和ML93355_c1(氨基環(huán)丙烷羧酸合成酶,ACS)在根瘤形成不同時期被強(qiáng)烈誘導(dǎo),銅脅迫下,ML98991_c3主要在結(jié)瘤晚期被抑制,而ML93355_c1在接菌后10,30d被抑制;兩個乙烯受體基因(ML46877_c0和ML84940_c2)和一個EIN3靶向F盒基因(ML46356_c0)分別在結(jié)瘤中后期明顯上調(diào),銅脅迫下,ML84940_c2和ML46356_c0在結(jié)瘤過程中受到不同程度的誘導(dǎo),但是ML46877_c0在結(jié)瘤晚期被抑制;此外,5個AP2/ERF家族基因ML102013_c0、ML99097_c0、ML99511_c0、ML85546_c0、ML98388_c0在接種后30d的根和根瘤中都有表達(dá),除了ML99097_c0,其余4個基因的表達(dá)在共生根及根瘤中被抑制。3.銅離子結(jié)合及轉(zhuǎn)運相關(guān)基因的表達(dá)分析表明,所檢測的三個基因ML98423_c0、ML52550_c0和ML98670_c0分別編碼Cu轉(zhuǎn)運ATP酶、高親和性銅轉(zhuǎn)運蛋白及推測的重金屬轉(zhuǎn)運/脫毒超家族蛋白成員,在Cu脅迫條件下,ML98670_c0、ML52550_c0表達(dá)下調(diào),ML98423_c0表達(dá)上調(diào),可能和Cu脅迫條件下維持植物體內(nèi)的銅離子穩(wěn)態(tài)平衡有關(guān)。接菌后這3個基因表達(dá)上調(diào),特別是ML52550_c0和ML98670_c0上調(diào)明顯,表明它們可能參與了根瘤形成的調(diào)控。此外,兩個吞噬細(xì)胞相關(guān)基因ML85004_c0、ML89530_c0在根和根瘤中有著不同的表達(dá)。4.RNAi結(jié)果顯示,Cu33611沉默后天藍(lán)苜蓿表現(xiàn)出植株矮小,結(jié)瘤數(shù)減少,根瘤較小;接菌10d的侵染事件分析表明,和對照相比,干擾后根毛卷曲,侵染線數(shù)目以及根瘤原基數(shù)目都顯著降低;接菌后30d的根瘤石蠟切片顯示,干擾后的天藍(lán)苜蓿植株根瘤中的侵染細(xì)胞在數(shù)目及密集程度上都遠(yuǎn)低于對照根瘤,侵染區(qū)和固氮區(qū)的侵染細(xì)胞都減少,結(jié)瘤明顯受到影響。這些結(jié)果表明,Cu33611確實參與了結(jié)瘤過程的調(diào)控,其具體功能還需要進(jìn)一步研究。
[Abstract]:The effects of heavy metal stress on the symbiotic nitrogen fixation are mostly in phenotypic observation and physiological data analysis, and there are few reports on the molecular mechanism of heavy metals. In this study, some genes expressed in the common roots of Alfalfa under copper stress separated by RNA-seq in the early laboratory were used as the research object. The expression analysis of copper stress and symbiotic process was carried out to detect their relationship with copper stress and symbiosis. At the same time, RNA interference technique was used to identify a chitinase encoding gene Cu33611, which was related to copper stress and specifically expressed in mycorrhiza. The content and results of the study were carried out. The following are as follows: 1. the cysteine cluster protein (CCP) gene, which is rich in cysteine rich clusters, is considered to be associated with defense and symbiosis. 9 CCP genes are selected for the expression analysis of the symbiotic process. The expression of 9 CCP genes can be broadly divided into three categories: the expression level of ML68936_c0 and ML94029_c0 is reached or close to the highest after receiving the bacteria. The level, then decreased, until the 30d returned to high levels, mainly in the early and late stages of the nodulation; ML67778_c0, ML85596_c0, ML98221_c0, ML101635_c0 and ML68801_c0 were higher in the middle of the nodule formation, and reached the highest level after the inoculation of the bacteria, and may play a role in the mid-term and late nodulation; ML54082_c0, ML64613_c0 and ML90197_c0 were At the late stage of the nodule formation, the expression was high and the other detection time points were not expressed or low level. It was presumed that the expression of.2. and ethylene synthesis and signal transduction related genes were mainly expressed in the late stage of the formation of the root nodules. The gene ML98991_c3 (aminopropane carboxylic oxidase, ACO) and ML93355_c1 (amino ring) related to ethylene synthesis were found. Propane carboxylic synthetase (ACS) was strongly induced at different stages of nodule formation. Under copper stress, ML98991_c3 was inhibited mainly in late nodulation, and 10,30d was suppressed after ML93355_c1; two ethylene receptor genes (ML46877_c0 and ML84940_c2) and a EIN3 target F box basis (ML46356_c0) were significantly up-regulated in the late nodulation, copper coercion. ML84940_c2 and ML46356_c0 were induced to varying degrees in the nodulation process, but ML46877_c0 was suppressed in the late nodulation; in addition, 5 AP2/ERF family genes, ML102013_c0, ML99097_c0, ML99511_c0, ML85546_c0, and ML98388_c0 were expressed in the roots and nodules of 30d after inoculation, and the expression of the other 4 genes was symbiotic except for ML99097_c0. The expression of.3. copper ion binding and transport related genes in root and root nodules showed that the three detected genes, ML98423_c0, ML52550_c0 and ML98670_c0, encoded Cu transport ATP, high affinity copper transporters and speculating heavy metal transport / detoxification superfamily proteins, ML98670_c0, ML52550_c0 table under Cu stress. Down regulation, up regulation of ML98423_c0 expression, may be related to the homeostasis of copper ions in the plant under Cu stress. These 3 genes are up regulated, especially the up regulation of ML52550_c0 and ML98670_c0, indicating that they may be involved in the regulation of the formation of nodules. In addition, two phagocyte related genes, ML85004_c0, ML89530_c0, The results of different expressions of.4.RNAi in root and root nodules showed that after Cu33611 silencing, alfalfa showed small plants, fewer nodules and smaller root nodules. The analysis of infection events of 10d of 10d showed that the root hair curl, the number of infection lines and the primary cardinal number of the root nodules were significantly lower than those of the control; the paraffin section of 30d after the inoculation was sliced. The results showed that the number and density of the infected cells in the rhizoma Rhizoma of alfalfa were much lower than that of the control root nodules, and the infected cells in the infected area and the nitrogen fixing area were reduced, and the nodulation was obviously affected. These results showed that Cu33611 did participate in the regulation of the nodulation process, and the specific functions needed to be further studied.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

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