铜胁迫下天蓝苜蓿共生根中差异基因的表达分析及功能鉴定

发布时间：2018-04-30 23:33

本文选题：天蓝苜蓿 + 铜胁迫　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：重金属胁迫对共生固氮的影响,目前多停留在表型观察及生理数据分析,对其分子机制方面的研究鲜有报道。本研究以豆科植物天蓝苜蓿作为研究对象,对实验室前期通过RNA-seq分离到的铜胁迫下天蓝苜蓿共生根中差异表达的部分基因,分别进行铜胁迫及共生过程的表达分析,检测它们与铜胁迫及共生的关系;同时,利用RNA干扰技术,对前期验证过的一个与铜胁迫相关且在接菌根中特异表达的几丁质酶编码基因Cu33611,进行结瘤过程的功能鉴定。研究内容及得到的结果简述如下:1.编码富含半胱氨酸簇的蛋白质(cysteine cluster protein,CCP)基因被认为与防御和共生相关。选择9个CCP基因进行共生过程的表达分析,9个CCP基因的表达大体上可分为三类:ML68936_c0和ML94029_c0的表达水平在接菌后7d达到或接近最高水平,随后下降,直至接菌后30d恢复高水平表达,主要在结瘤的早期和晚期表达;ML67778_c0、ML85596_c0、ML98221_c0、ML101635_c0和ML68801_c0在根瘤形成中期表达量较高,接菌后30d时达到最高,可能在结瘤中期和晚期起作用;ML54082_c0、ML64613_c0和ML90197_c0则在根瘤形成晚期表达量较高,其他检测时间点不表达或低水平表达,推测其主要在根瘤形成晚期发挥作用。2.与乙烯合成及信号转导相关基因的表达分析显示,乙烯合成有关的基因ML98991_c3(氨基环丙烷羧酸氧化酶,ACO)和ML93355_c1(氨基环丙烷羧酸合成酶,ACS)在根瘤形成不同时期被强烈诱导,铜胁迫下,ML98991_c3主要在结瘤晚期被抑制,而ML93355_c1在接菌后10,30d被抑制;两个乙烯受体基因(ML46877_c0和ML84940_c2)和一个EIN3靶向F盒基因(ML46356_c0)分别在结瘤中后期明显上调,铜胁迫下,ML84940_c2和ML46356_c0在结瘤过程中受到不同程度的诱导,但是ML46877_c0在结瘤晚期被抑制;此外,5个AP2/ERF家族基因ML102013_c0、ML99097_c0、ML99511_c0、ML85546_c0、ML98388_c0在接种后30d的根和根瘤中都有表达,除了ML99097_c0,其余4个基因的表达在共生根及根瘤中被抑制。3.铜离子结合及转运相关基因的表达分析表明,所检测的三个基因ML98423_c0、ML52550_c0和ML98670_c0分别编码Cu转运ATP酶、高亲和性铜转运蛋白及推测的重金属转运/脱毒超家族蛋白成员,在Cu胁迫条件下,ML98670_c0、ML52550_c0表达下调,ML98423_c0表达上调,可能和Cu胁迫条件下维持植物体内的铜离子稳态平衡有关。接菌后这3个基因表达上调,特别是ML52550_c0和ML98670_c0上调明显,表明它们可能参与了根瘤形成的调控。此外,两个吞噬细胞相关基因ML85004_c0、ML89530_c0在根和根瘤中有着不同的表达。4.RNAi结果显示,Cu33611沉默后天蓝苜蓿表现出植株矮小,结瘤数减少,根瘤较小;接菌10d的侵染事件分析表明,和对照相比,干扰后根毛卷曲,侵染线数目以及根瘤原基数目都显著降低;接菌后30d的根瘤石蜡切片显示,干扰后的天蓝苜蓿植株根瘤中的侵染细胞在数目及密集程度上都远低于对照根瘤,侵染区和固氮区的侵染细胞都减少,结瘤明显受到影响。这些结果表明,Cu33611确实参与了结瘤过程的调控,其具体功能还需要进一步研究。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

【相似文献】