玉米NAC膜结合转录因子基因克隆及表达调控研究

发布时间：2018-05-27 05:00

本文选题：玉米 + NAC膜结合转录因子　；参考：《山东农业大学》2016年博士论文

【摘要】：转录因子在调控植物发育以及抗逆功能基因的表达中具有重要作用,其中,NAC转录因子是植物特有的转录调控因子,在植物的器官发育、形态建成、器官分离、叶片衰老以及非生物胁迫响应中发挥重要的调控作用。膜结合转录因子是一类特殊的转录因子,通常情况下,它以膜结合的形式存在于细胞的膜结构(细胞膜、内质网膜或核膜)上,处于非活性状态。在受到外界信号刺激后,通过蛋白水解机制从膜上释放下来,转变为有活性的形式进入细胞核发挥作用。目前,有关玉米NAC膜结合转录因子(ZmNTLs)的功能研究尚未见报道。本研究从玉米基因组中,发掘出了7个ZmNTLs基因,对它们的蛋白结构特点、系统进化树、亚细胞定位、转录激活活性、组织特异性表达及其对非生物胁迫及激素响应特性进行了研究,获得了拟南芥35S::ZmNTL1、35S::ZmNTL2和35S::ZmNTL5纯系,并分别研究了ZmNTL1,-2在调控拟南芥水淹胁迫响应和木质素合成中的作用。主要研究过程及实验结果包括:(1)玉米NAC膜结合转录因子ZmNTLs序列、表达特征分析利用生物信息学方法,从216个玉米NAC转录因子中筛选出7个具有跨膜结构域的成员,确定为玉米NAC膜结合转录因子(ZmNTL),并命名为ZmNTL1-7。序列比对发现,所有的玉米NTLs N-末端均具有典型的NAC结构域,C-末端均具有1个跨膜螺旋结构域。氨基酸序列比对,将7个ZmNTLs分成4个组(包括3个基因对),分别为ZmNTL1/5、ZmNTL2/3、ZmNTL6/7和ZmNTL4。(2)ZmNTLs进化分析利用MEGA6.0对来自拟南芥的18个NTLs蛋白、水稻的5个NTLs蛋白、谷子的8个NTLs蛋白同玉米的7个NTL蛋白进行了进化树构建,结果表明上述38个膜结合转录因子被分成5个亚组,ZmNTLs分布于其中的I-IV 4个亚组中,ZmNTL2/ZmNTL3和At NTL4位于第I组内,ZmNTL4和At NTL7位于第II组内,ZmNTL1/ZmNTL5、At NTL8和At NTL5位于第III组内,ZmNTL6/ZmNTL7和At NTL6和At NTL9位于第IV组内,预测在一个亚组中的成员可能有相似功能。通过MEME进行的保守motif分析表明,玉米NTLs的保守motif比拟南芥多。ZmNTLs与At NACs、Os NACs的进化关系分析发现,ZmNTLs与拟南芥中的某些NAC转录因子具有较近的进化关系,例如ZmNTL1与ANAC086、ANAC045,ZmNTL2与VND6、VND7等,这预示着ZmNTLs可能与某些拟南芥的NAC有相似的功能。(3)ZmNTLs亚细胞定位及转录激活活性分析以玉米B73自交系为材料,克隆了7个ZmNTLs基因的CDS,构建了它们的GFP瞬时表达载体,将它们转化到拟南芥原生质体中,发现ZmNTL1-5和7定位于细胞质膜上;ZmNTL6定位于内质网膜上。酵母转录激活活性分析发现,所有的ZmNTLs均有转录激活活性。β-半乳糖苷酶报告基因定量检测表明ZmNTL2转录激活活性最高,其次是ZmNTL6;ZmNTL4与ZmNTL7相对较弱。(4)ZmNTLs的时空表达模式Real-time PCR分析表明,ZmNTL1-7表达具有明显的组织特异性。ZmNTL1,-2在根、茎、叶中均有表达,其中ZmNTL1在叶中表达量最高,ZmNTL2在茎中表达量最高,ZmNTL3-7主要在茎中表达。(5)ZmNTLs对非生物胁迫及激素的响应模式对三叶期玉米幼苗进行了非生物胁迫(干旱、高盐、低温、H2O2)及激素(ABA、BR、GA、2,4-D、Me JA、KT)处理下的Real-time PCR分析,结果表明ZmNTL1-7对非生物胁迫及激素响应的表达模式表现出多样性特征。在ABA、H2O2胁迫下,ZmNTL1-7在根和茎中均显著上调,表达模式相近;而在PEG处理下,ZmNTL1-7在茎中表达显著下调;ZmNTL1-7均受ABA、Me JA、BR、2,4-D、KT诱导为上调表达;尤其对ABA和Me JA响应最为强烈;GA处理下表达最弱,ZmNTL1,-4,-6有少量诱导,ZmNTL7被抑制,ZmNTL2,-3,-5没有明显变化。(6)玉米ZmNTL1、ZmNTL2、ZmNTL5拟南芥过表达系的获得将ZmNTL1、ZmNTL2、ZmNTL5全长及其去除跨膜结构域(△TM)的片段连接到过表达载体p K2GW7中,分别获得了它们的转基因拟南芥纯系种子。发现与在正常生长条件下,它们的主根长度及苗生长势与野生型无差异。(7)ZmNTL拟南芥过表达系对过氧化氢胁迫的响应ZmNTL1、-2、-5去除跨膜结构域(△TM)的转基因拟南芥过表达系野生型(Col-0)的种子分别点在1/2MS固体培养基上,根长到1cm左右时,转移到含有0.5mM、1mM、1.5mM和2mM H2O2的培养基上进行胁迫处理,结果表明转基因系能够明显缓解了过氧化氢的抑制作用,这三个ZmNTLs基因都响应过氧化氢胁迫。(8)ZmNTL1在水淹胁迫中的作用研究在土壤水分饱和的状态下,35S::ZmNTL1转基因拟南芥生长状态与对照无明显差异,35S::ZmNTL1-△TM生长状态明显弱于对照,表现出发育迟缓、植株矮化、叶片卷曲,表明ZmNTL1降低了拟南芥对水淹胁迫的耐受性。Real-time PCR分析表明,在水淹下,35S::ZmNTL1-△TM中的At Rrboh D表达明显低于对照,At ICL、NADP-ME1、At ADH1表达显著高于对照。EMAS(凝胶阻滞实验)分析发现,ZmNTL1-△TM蛋白能够与At Rboh D启动子的4个位点结合,表明ZmNTL1直接调控At Rboh D的表达。发现在水淹下,拟南芥rbohd(敲除系)生长受到抑制,而35S::At Rrboh D生长优于对照,表明At Rrboh D参与了拟南芥对水淹胁迫的调控。将At Rboh D的过表达载体转入35S::ZmNTL1-△TM中,35S::ZmNTL1-△TM对水淹的敏感性得到恢复,从遗传上证明了ZmNTL1和At Rboh D的直接调控关系。综上,ZmNTL1作为一个水淹胁迫的负调控因子,可通过直接下调At Rboh D表达来降低对水淹的耐受性。(9)ZmNTL2在调控植物木质素合成中的作用研究35S::ZmNTL2和35S::ZmNTL1-△TM转基因拟南芥生长7周时,茎的硬度明显优于野生型。甲苯胺蓝和间苯三酚染色发现,与野生型相比,过表达系植株茎木质部中被染色的细胞数明显增多,且着色深,表明木质素分布范围和含量均高于对照。原位杂交结果表明,ZmNTL2主要在木质部表达。Real-time PCR分析表明,木质素合成的关键基因PAL、C4H、HCT、CAD、CCR、C3H、COMT、F5H在35S::ZmNTL2过表达系和35S::ZmNTL1-△TM过表达系中明显上调。综上,ZmNTL2可能通过上调木质素合成相关基因表达来提高植株茎的木质化程度。总之,玉米ZmNTL家族成员基因在植物发育和响应非生物胁迫中起着重要作用,挖掘ZmNTLs基因的功能对解析玉米生长发育及耐逆性作用机制有重要意义。
[Abstract]:Transcription factors play an important role in regulating plant development and the expression of anti inverse function genes. NAC transcription factors are specific regulatory factors of plants, which play important roles in plant organ development, morphogenesis, organ separation, leaf senescence and abiotic stress response. Membrane binding transcription factors are a class of transcription factors. A special transcription factor, usually in the form of membrane binding that exists in the membrane structure of the cell (cell membrane, endoplasmic reticulum or nuclear membrane) and is in an inactive state. After being stimulated by external signals, it is released from the membrane through the proteolysis mechanism and transformed into active form into the nucleus. The functional study of NAC membrane binding transcription factor (ZmNTLs) has not yet been reported. In this study, 7 ZmNTLs genes were unearthed from the maize genome. Their protein structure characteristics, phylogenetic tree, subcellular location, transcriptional activation activity, tissue specific expression, and characteristics of abiotic stress and hormone response were studied. 35S:: ZmNTL1,35S:: 35S:: ZmNTL1,35S:: ZmNTL2 and 35S:: ZmNTL5 pure lines, and the role of ZmNTL1 and -2 in regulating water flooding stress response and lignin synthesis in Arabidopsis, respectively. The main research process and experimental results include: (1) ZmNTLs sequence of transcription factor in corn NAC membrane, expression characteristics analysis using bioinformatics method, from 216 corn NAC to NAC 7 members with transmembrane domain were selected and identified as NAC membrane binding transcription factor (ZmNTL), and named as ZmNTL1-7. sequence alignment. All maize NTLs N- ends have typical NAC domain, and all C- ends have 1 transmembrane spiral domains. Amino acid sequence alignment and 7 ZmNTLs are divided into 4 groups (packages). Including 3 gene pairs, ZmNTL1/5, ZmNTL2/3, ZmNTL6/7 and ZmNTL4. (2) ZmNTLs evolution analysis using MEGA6.0 to 18 NTLs proteins from Arabidopsis, 5 NTLs proteins of rice, 8 NTLs protein of millet and 7 NTL proteins of maize, the results showed that the above 38 membrane binding transcription factors were divided into 5 subgroups, ZmNT Ls is distributed in the 4 subgroups of I-IV, and ZmNTL2/ZmNTL3 and At NTL4 are located in group I, ZmNTL4 and At NTL7 are in group II. The analysis showed that the conservative motif of maize NTLs compared the evolutionary relationship between.ZmNTLs and At NACs, Os NACs, and found that ZmNTLs and some NAC transcriptional factors in Arabidopsis have closer evolutionary relationships, such as ZmNTL1 and ANAC086, ANAC045, etc., which may have similar functions to some Arabidopsis. 3) ZmNTLs subcellular localization and transcriptional activation activity analysis using maize B73 inbred line as material, cloned 7 ZmNTLs genes CDS, constructed their GFP transient expression vector, transformed them into Arabidopsis protoplasts, and found ZmNTL1-5 and 7 located on the cytoplasmic membrane; ZmNTL6 was located on the endoplasmic reticulum. Yeast activation active fraction was determined. It was found that all ZmNTLs had transcriptional activation activity. Quantitative detection of beta galactosidase reporter gene showed that ZmNTL2 transcriptional activation activity was the highest, followed by ZmNTL6; ZmNTL4 and ZmNTL7 were relatively weak. (4) Real-time PCR analysis of ZmNTLs's spatio-temporal expression pattern showed that ZmNTL1-7 expression had obvious tissue specific.ZmNTL1, -2 in roots, stems and leaves. The expression of ZmNTL1 was the highest in the leaves, the expression of ZmNTL2 in stem was the highest, and ZmNTL3-7 was mainly expressed in the stem. (5) ZmNTLs was used for abiotic stress (drought, high salt, low temperature, H2O2) and hormone (ABA, BR, GA, 2,4-D, Me JA, KT) in the three leaf period maize seedlings. R analysis showed that ZmNTL1-7 showed diversity in the expression patterns of abiotic stress and hormone response. Under the stress of ABA and H2O2, ZmNTL1-7 was significantly up-regulated in the root and stem, and the expression pattern was similar, while ZmNTL1-7 was significantly down regulated in the stem under PEG treatment; ZmNTL1-7 was induced to be up expression by ABA, Me JA, BR. The response to ABA and Me JA was most intense; GA treatment was the weakest, ZmNTL1, -4, -6 had a small amount of induction, ZmNTL7 was suppressed, ZmNTL2, -3, -5 had no obvious changes. The homozygous seeds of transgenic Arabidopsis were obtained. It was found that the length of the main root and the growth potential of the transgenic Arabidopsis were not different from those of the wild type. (7) the response of the overexpression line of ZmNTL Arabidopsis to ZmNTL1, -2, and -5 to remove the transmembrane domain (delta TM) of the transgenic Arabidopsis overexpressed line (Col-0) On the 1/2MS solid medium, the seeds were transferred to the medium containing 0.5mM, 1mM, 1.5mM and 2mM H2O2 on the medium of 1cm, respectively. The results showed that the transgenic lines could significantly alleviate the inhibition of hydrogen peroxide, and the three ZmNTLs genes were responsive to hydrogen peroxide stress. (8) the effect of ZmNTL1 on water flooding stress Under the condition of soil water saturation, the growth state of 35S:: ZmNTL1 transgenic Arabidopsis was not significantly different from that of the control. The growth state of 35S:: ZmNTL1- Delta TM was obviously weaker than that of the control. The growth retardation, the dwarf plant and the curl of leaves showed that ZmNTL1 decreased the tolerance.Real-time PCR analysis of Arabidopsis to water flooding stress, which showed that the water flooded, under water flooding, 35S:: the expression of At Rrboh D in ZmNTL1- Delta TM was significantly lower than that of the control. The expression of At ICL, NADP-ME1, At ADH1 was significantly higher than that of the control.EMAS (gel block experiment). The growth of 35S:: At Rrboh D is better than that of the control, which indicates that At Rrboh D participates in the regulation of water flooding stress in Arabidopsis thaliana. The over expression vector of At Rboh D is transferred to 35S:: ZmNTL1- Delta, the sensitivity to water flooding is restored. For a negative regulator of water flooding stress, the tolerance to water flooding can be reduced by direct downregulation of At Rboh D. (9) the effect of ZmNTL2 on the regulation of lignin synthesis in plants: 35S:: ZmNTL2 and 35S:: ZmNTL1- Delta and TM transgenic Arabidopsis thaliana growth at 7 weeks, the stem hardness is better than that of wild type. Toluidine blue and benzol three phenol are dyed. Compared with the wild type, the number of stained cells in the xylem of the overexpressed plant increased obviously, and the coloring was deep, indicating that the distribution and content of lignin were higher than those of the control. The results of in situ hybridization showed that ZmNTL2 was mainly expressed in xylem by.Real-time PCR analysis, which indicated that the key genes of lignin synthesis, PAL, C4H, HCT, CAD, CCR, C3H, COMT, F5H. In 35S:: ZmNTL2 overexpression line and 35S:: ZmNTL1- Delta TM overexpression line obviously up-regulated. To sum up, ZmNTL2 may improve the lignification degree of plant stem by up regulation of lignin related gene expression. In a word, the gene of maize ZmNTL family members plays an important role in plant development and response to abiotic coercion, mining the function of ZmNTLs gene. It is important to understand the mechanism of maize growth and development and stress tolerance.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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