香蕉SOD基因家族的全基因组鉴定及功能分析

发布时间：2018-01-02 22:22

  本文关键词：香蕉SOD基因家族的全基因组鉴定及功能分析　出处：《福建农林大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 香蕉 SOD基因家族 启动子 非生物胁迫 激素处理 表达分析

【摘要】：香蕉是热带和亚热带地区重要的经济和粮食作物。但在生产上,易受冬春季低温和夏季干旱等不利环境的影响,而导致植株生长发育受阻或减产,给蕉农造成较大经济损失。逆境胁迫往往诱导植物细胞内活性氧自由基的过量积累,进而产生氧化胁迫,影响植物的生长发育和结果。超氧化物歧化酶(Superoxide dismutase,SOD)是抗氧化系统的第一道防线,能有效的清除活性氧并减轻氧化胁迫从而提高植物对逆境的耐受能力。已有研究表明香蕉SOD同工酶谱和酶活性在低温等非生物胁迫下发生显著改变,说明SOD与香蕉抗逆境过程密切相关。但目前对香蕉SOD基因及其表达的调控机制还知之甚少。因此,本研究以两个野生蕉的全基因组序列为参考,在栽培香蕉中对SOD基因家族进行系统的克隆鉴定,分析不同成员的启动子序列和顺式调控元件,对不同SOD基因成员在非生物胁迫和激素处理下的表达调控进行研究,以探讨SOD在香蕉抗逆过程中的作用。主要研究结果如下：1.香蕉SO1)基因家族的全基因组分析与克隆①采用计算机分析方法从小果野蕉DH-Pahang(Musa acuminata,AA genome)的全基因组中共检索到15条SOD候选序列,其中2条为冗余序列。从野蕉PKW(Musa balbisiana,BB genome)的全基因组中共检索到14条SOD候选序列,其中1条为冗余序列,2条为嵌合基因。②根据两个野生蕉的SOD序列设计引物,以福建天宝蕉(Cavendish banana,AAA genome)为材料,共克隆得到25条不同的SOD mRNA转录本。它们分别由6个Cu/ZnSOD基因、4个MnSOD基因和2个FeSOD基因转录。可变剪接、可变转录起始位点和3'UTR选择性多聚腺苷酸化是导致MaSOD基因mRNA多样性的原因。③以DNA为模板,克隆了12个MaSOD家族基因的gDNA序列,大小为1807～4720 bp。根据基因结构不同分为4组：MaCSD1A、MaCSD1B、MaCSD1C和MaCSD1L共4个成员为Ia组,均含有6个内含子。MaCSD2A和MaCSD2B构成Ib组,均含有7个内含子。4个MnSOD (MaMSD1A、MaMSD1B、MaMSD1C和MaMSD1D)组成II组,均含有5个内含子。III组包含MaFSD1A和MaFSD11共2个成员,但它们的外显子数目存在差异；MaFSD1A含有7个内含子,而MaFSD1B含有8个内含子。该分类结果与MaSOD家族基因氨基酸序列的聚类结果及根据它们保守基序的分类结果一致。④物种内和物种间的共线性分析结果表明,全基因组复制和染色体区段复制是香蕉SOD基因家族数量扩张的主要因素。⑤对不同基因组类型香蕉SOD家族基因的比较分析结果表明,福建天宝蕉(AAA genome)的SOD基因与福州野生蕉(AA genome)和小果野蕉DH-Pahang(AA genome)的SOD基因有较高的一致性,但与野蕉PKW (BB genome)的SOD基因的一致性较低。2.香蕉SOD基因家族编码蛋白的生物信息学分析根据生物信息学分析结果,香蕉SOD家族基因编码的蛋白与其他植物SOD具有很高的同源性,且都含有SOD保守结构域和特征氨基酸位点,说明他们在进化上较为保守,在功能上具有相似性。但不同MaSOD蛋白仍存在各自的特点。MaSOD蛋白家族的分子量大小在15037.6～34235.5 Da,其中FeSOD的分子量最大,MnSOD的分子量次之,Cu/ZnSOD的分子量最小。除了MaMSD1C为碱性蛋白,MaCSD2B 和 MaMSDIA为弱碱性蛋白外,其余的成员均为酸性蛋白。除了MaFSD1A为不稳定蛋白外,其余的11个成员均为稳定蛋白。除了MaCSD2A 和 MaCSD2B为疏水性蛋白外,其余的均为亲水性蛋白。MaCSD1的4个蛋白(MaCSD1A、MaCSDIB、MaCSD1C 和 MaCSD1D)由19种氨基酸组成,都不含有色氨酸；其余的MaSOD家族蛋白均由20种氨基酸组成。亚细胞定位分析显示4个MaCSD1蛋白(MaCSD1A、MaCSD1B、MaCSD1C和MaCSD1D)定位于细胞质；2个MaCSD2蛋白(MaCSD2A 和 MaCSD2B)定位于叶绿体；4个MaMSD蛋白(MaMSD1A、MaMSD1B、 MaMSD1C 和 MaMSD1D)定位于线粒体；而MaFSD1A主要定位于叶绿体,在细胞质中也存在,MaFSD1B则主要定位于叶绿体。磷酸化位点预测分析结果表明不同MaSOD蛋白成员间的各种磷酸化位点的数量和位置存在明显差异,说明香蕉MaSOD家族的不同蛋白成员可能在翻译后水平受不同的磷酸化方式调控表达。3.香蕉SOD基因家族启动子的克隆与分析采用PCR法直接克隆得到MaSOD家族的11条大小在1084～2114 bp的5’端调控序列。序列分析显示,它们不仅包含核心启动子区和启动子核心元件TATA-box 和 CAAT-box,还含有大量与光响应、环境胁迫应答、激素响应和生理节律调控等相关的顺式元件。对MaSOD基因家族不同成员的启动子顺式元件的比较分析表明,不同成员的启动子间除了都含有各自特异的顺式元件外,还具有一些共有的顺式元件以及响应同一胁迫的不同顺式元件。说明在一定程度上,同一胁迫可以调控多个香蕉SOD成员的表达,但各个成员在响应同一胁迫上又具有一定的差异性。4.香蕉SOD基因家族在非生物胁迫和激素处理下的表达分析qRT-PCR法分析MaSOD基因家族在不同组织部位的表达情况,结果表明除了MaCSD2B基因在假茎中未检测到表达外,其余的11个成员在叶、假茎和根中都有表达。qRT-PCR法分析MaSOD基因家族在低温、高温、干旱和NaCL胁迫下的表达模式。结果表明香蕉MaSOD基因在不同非生物胁迫下,并非是简单的一对一响应,而是存在较为复杂的综合响应。具体情况如下：在低温胁迫48 h时,香蕉MaCSD2A基因显著上调表达,而MaCSD1D和MaCSD2B则显著下调表达,其余成员的表达差异不明显。在高温胁迫48 h时,有6个基因(MaCSD1B、MaCSD1D、MaMSD1A、MaMSD1B、MaMsD1C和MaFSD1A)呈现显著上调表达,只有MaCSD2A基因在12h时显著下调表达。而在干旱胁迫下,只有3个Cu/ZnSOD基因(MaCSD1B、MaCSD1C和MaCSD2A)在不同的时间点显著上调表达；其余的成员多数表达量呈现下调,其中5个成员(MaCSD1A、MaCSD1D、MaMSD1A、 MaMSD1B和MaFSD1B)表达量的下调倍数达2～10倍。高盐胁迫显著诱导MaCSD1D、 MaMSDIA和MaMSD1B基因的表达,但抑制MaCSD2A和MaFSD1B基因的表达。qRT-PC R法分析MaSOD基因家族在脱落酸、赤霉素、生长素和水杨酸4种激素处理下的表达模式。结果表明只有MaCSD和MaMSD亚家族的成员被显著诱导表达,而MaFSD亚家族的成员表达量变化不明显。具体情况如下：在脱落酸处理下,MaCSD1D和MaMSD1A被诱导显著上调表达。在赤霉素处理后,也仅有2个基因(MaCSD1D和MaMSD1A)在不同时间点显著上调表达,其他成员的表达差异不显著。生长素处理则可诱导3个MaCSD基因(MaCSDIA、MaCSD1D和MaCSD2B)和1个MaMSD基因(MaMSD1A)在不同时间点显著上调表达。而水杨酸处理24 h时显著诱导MaCSD1D的上调表达。5.香蕉铜锌超氧化物歧化酶MaCSD1D基因的功能分析细胞质型的MaCSD1D基因在非生物胁迫(冷、热、干旱和盐胁迫)和激素处理(ABA、GA3、IAA和SA)下均有显著的上调或下调表达,暗示该基因可能在香蕉的逆境胁迫中发挥重要作用。为了进一步揭示其功能,构建MaCSD1D基因的过表达载体并转化烟草,对MaCSD1D基因在低温胁迫下的功能进行分析,结果表明转MaCSD1D基因的烟草种子在低温下的萌发速度和生长状况均优于非转基因烟草的种子。6片功能叶时期的转基因烟草比非转基因烟草对低温胁迫表现出更强的耐受能力,且转基因烟草在低温下的SOD酶活性高于非转基因烟草的SOD酶活性。6.香蕉Cu/ZnSOD分子伴侣蛋白基因MaCCS的克隆与表达分析铜锌超氧化物歧化酶(Cu/ZnSOD)是一种同时含铜和锌的金属酶,在正常生理条件下铜的获取需要通过分子伴侣蛋白CCS来实现。本研究采用RT-PCR结合RACE-PCR获得了1条新的CCS基因(MaCCS)。序列分析表明MaCCS具有典型的CCS结构域和保守的基因结构。MaCCS基因启动子上存在大量参与非生物胁迫和激素应答的顺式元件。qRT-PCR分析表明MaCCS基因在叶、假茎和根部均有表达,并参与非生物胁迫(CuS04、热、冷和干旱)和激素(ABA和IAA)应答。而且,MaCCS基因的转录模式在低温胁迫下与MaCSD1B、 MaCSD1D和MaCSD2B基因的相似,在热胁迫下与MaCSD1B和MaCSD1D基因的相似,在干旱胁迫下与MaCSD1B和MaCSD1C基因的相似,在ABA和IAA处理下则与MaCSD1A、 MaCSD1C和MaCSD2B基因的相似。这说明在非生物胁迫和激素处理下,香蕉MaCCS基因在转录水平的表达与其对应的MaCSD基因的表达呈现正相关,暗示MaCCS基因可能与MaCSD基因协作参与香蕉的抗逆过程。
[Abstract]:Banana is economy and an important food crop in tropical and subtropical regions. But in production, is easily affected by low temperature in winter and spring and summer drought and other adverse environment, resulting in plant growth and development is blocked or reduced, resulting in greater economic losses to farmers. Excessive accumulation of stress often induced by active oxygen free radicals in plant cells. Resulting in oxidative stress affects plant growth and results. Superoxide dismutase (Superoxide dismutase SOD) is the first line of the antioxidant system, can effectively remove active oxygen and reduce oxidative stress and improve plant tolerance to adversity. Studies have shown that significant changes of banana SOD isozymes and enzyme activity occurred at low temperature under abiotic stress, closely related to SOD and banana process. But the anti stress regulation mechanism and expression of SOD gene in banana is poorly understood because. This, in this study, the whole genome sequence of two wild banana as a reference, cloning and identification system of SOD gene family in banana cultivation, analysis of promoter sequences and cis regulatory elements of different members, to study the different expression of SOD gene in members of abiotic stress and hormone treatment under control, to investigate the SOD the resistance function in the process of banana. The main results are as follows: 1.) banana SO1 whole genome analysis and cloning of the gene family using computer analysis method from the wild banana (Musa DH-Pahang fruit acuminata, AA genome) genome were searched and the 15 SOD sequences, 2 of which were from redundant sequences. Musa PKW (Musa balbisiana, BB genome) of the whole genome retrieved 14 SOD candidate sequences, 1 of which are redundant sequences, 2 chimeric genes. The primers were designed according to SOD sequence of two wild banana, with good fortune Tianbao banana (Cavendish banana, built AAA genome) as material, were cloned from 25 different SOD mRNA transcripts. They were composed of 6 Cu/ZnSOD genes, 4 MnSOD genes and 2 FeSOD genes transcription. Alternative splicing, variable transcription initiation sites and 3'UTR selective polyadenylation of MaSOD gene is the cause of mRNA diversity. With DNA as the template, and cloned the gDNA sequence of 12 MaSOD genes, the size of 1807 ~ 4720 bp. according to the gene structure were divided into 4 groups: MaCSD1A, MaCSD1B, MaCSD1C and MaCSD1L a total of 4 members of the Ia group, containing 6 introns of.MaCSD2A and MaCSD2B in Ib group. Contains 7 introns of.4 MnSOD (MaMSD1A, MaMSD1B, MaMSD1C and MaMSD1D). Group II contained 5 introns of MaFSD1A and MaFSD11 in.III group contains a total of 2 members, but their exon number differences; MaFSD1A contains 7 introns, while containing MaFSD1B There are 8 introns. The classification results of the clustering result and the amino acid sequence of the MaSOD gene family and their classification results based on the conserved motif. The collinearity within and between species analysis showed that the genome copy number and chromosomal regions are the main factors of banana SOD gene family. The genome of different expansion the type of banana SOD gene family analysis results show that Fujian Tianbao banana (AAA genome) SOD gene and Fuzhou wild banana (AA genome) and Musa acuminata (AA DH-Pahang genome) SOD gene has a high consistency, but with the wild banana (BB PKW genome) according to the analysis results of bioinformatics analysis the biological information consistency of the SOD gene of low.2. gene family encoding protein SOD banana, banana SOD gene family encoding protein with other plant SOD has high homology, which contains the conserved SOD. The domain and the characteristics of amino acid sites, indicating that they are more conservative in evolution, is similar in function. But the MaSOD protein still exist in molecular characteristics of.MaSOD protein family size in 15037.6 ~ 34235.5 Da, of which the molecular weight of FeSOD, the molecular weight of MnSOD times, the molecular weight of Cu/ZnSOD is the minimum. In addition to MaMSD1C for MaCSD2B and MaMSDIA as the basic protein, alkaline protein, the rest of the members are acidic protein. In addition to MaFSD1A for unstable protein, 11 members of the rest are stable. In addition to MaCSD2A and MaCSD2B protein is a hydrophobic protein, the rest are 4 hydrophilic protein.MaCSD1 (protein MaCSD1A, MaCSDIB, MaCSD1C and MaCSD1D) consisting of 19 kinds of amino acids, do not contain tryptophan; the rest of the MaSOD family proteins are composed of 20 amino acids. The subcellular localization analysis showed that the 4 MaCSD1 protein (MaCSD1A, MaC SD1B, MaCSD1C and MaCSD1D) located in the cytoplasm; 2 MaCSD2 proteins (MaCSD2A and MaCSD2B) located in the chloroplast; 4 MaMSD proteins (MaMSD1A, MaMSD1B, MaMSD1C and MaMSD1D) located in the mitochondria; while MaFSD1A is mainly located in the chloroplast, also exist in the cytoplasm, MaFSD1B was mainly located in the chloroplast. Phosphorylation sites prediction the analysis results show that there exist obvious differences in the number and location of various phosphorylation sites of MaSOD protein between the members of the MaSOD family, that banana different protein members may be on the translation level by different phosphorylation type expression cloning and analysis of.3. banana SOD gene family promoter by PCR directly cloned 11 size MaSOD the family in the 1084 ~ 2114 BP 5 'flanking sequence. Sequence analysis showed that they contain not only the core promoter TATA-box and CAAT-bo promoter and core promoter element X, also contains a large number and light response, response to environmental stress, hormone responses and circadian rhythm regulation and other related cis elements. The comparative analysis of different members of MaSOD gene family promoter cis element shows that different members of the promoter in addition to cis elements contain their own specific, also has some common some CIS elements and different cis elements in response to the same stress. To some extent, the same stress can regulate the expression of multiple banana SOD members, but each member in response to the same stress has the differential expression analysis of.4. banana SOD gene family in the abiotic stress and hormone treatment the qRT-PCR analysis of MaSOD gene family expression in different tissues, the results showed that the MaCSD2B gene in the stem was not detected in expression, the rest of the 11 members in the leaf, stem and root are false statement .qRT-PCR analysis of MaSOD gene family in low temperature, high temperature, drought and expression patterns under NaCL stress. The results showed that banana MaSOD gene in different abiotic stresses, is not a simple one to one response, but comprehensive response is more complex. The details are as follows: in the low temperature of 48 h, the expression of banana MaCSD2A the gene was significantly increased, while MaCSD1D and MaCSD2B were down regulated and expression of the remaining members of the high temperature stress is not obvious. In 48 h, 6 genes (MaCSD1B, MaCSD1D, MaMSD1A, MaMSD1B, MaMsD1C and MaFSD1A) showed significant upregulated, only significant down-regulation of MaCSD2A gene in 12h expression in drought. Only under the stress of 3 Cu/ZnSOD genes (MaCSD1B, MaCSD1C and MaCSD2A) expression at different time points increased significantly; the majority of the members expressed the rest of the downward, of which 5 members (MaCSD1A, MaCSD1D, MaMSD1A, MaMSD1 B and MaFSD1B) expression by 2 times to 10 times. The high salt stress significantly induced by MaCSD1D, expression of MaMSDIA and MaMSD1B genes, analysis of the MaSOD gene family in abscisic acid, gibberellin but inhibits the expression of.QRT-PC R MaCSD2A and MaFSD1B gene, the expression pattern of auxin and salicylic acid 4 kinds of hormone treatments. The results show that only members of the MaCSD and the MaMSD subfamily was significantly up-regulated, while the MaFSD subfamily expression did not change significantly. The details are as follows: in abscisic acid, MaCSD1D and MaMSD1A were significantly up-regulated in gibberellin induced. After treatment, only 2 genes (MaCSD1D and MaMSD1A) expression in different time the point was significantly increased, the other members of the expression difference was not significant. Auxin treatment can induce 3 MaCSD genes (MaCSDIA, MaCSD1D and MaCSD2B) and 1 MaMSD genes (MaMSD1A) at different time points was significantly up-regulated. The salicylic acid 24 h significantly induced the upregulation of MaCSD1D.5. analysis of banana copper zinc superoxide dismutase MaCSD1D gene function of MaCSD1D gene in the cytoplasm of stress type non biological expression (cold, heat, drought and salt stress) and hormone treatment (ABA, GA3, IAA and SA) expression were significantly upregulated or, suggesting that this gene may play an important role in banana stress. In order to further reveal the function, construction of over expression vector and transformed into tobacco MaCSD1D gene, the function of MaCSD1D gene under low temperature stress analysis, seed.6 leaves during the results show that MaCSD1D transgenic tobacco seeds at low temperature the speed of germination and growth were better than that of non transgenic tobacco and transgenic tobacco than non transgenic tobacco to low temperature stress showed stronger tolerance and SOD activity of transgenic tobacco under low temperature Cloning and expression of SOD is higher than that of.6. activity of banana Cu/ZnSOD molecular chaperone protein gene MaCCS of non transgenic tobacco analysis of copper zinc superoxide dismutase (Cu/ZnSOD) is a copper and zinc metal enzyme, under normal physiological conditions of copper acquisition needs to be realized through the molecular chaperone protein. This study used CCS RT-PCR combined with RACE-PCR obtained 1 new CCS gene (MaCCS). Sequence analysis showed that MaCCS is a typical CCS domain and the conserved gene structure of.MaCCS gene promoter in.QRT-PCR a large number of cis elements involved in abiotic stress and hormone response analysis showed that the MaCCS gene in leaves, stems and roots were false expression, and abiotic stress (CuS04, heat, cold and drought) and hormone (ABA and IAA) response. Moreover, the transcription pattern of MaCCS gene under low temperature stress and MaCSD1B, MaCSD1D and MaCSD2B genes were similar under heat stress With MaCSD1B and MaCSD1D genes of similar genes with MaCSD1B and MaCSD1C under drought stress was similar to that in ABA and IAA treatment with MaCSD1A, MaCSD1C and MaCSD2B genes are similar. This shows that in the abiotic stress and hormone treatments, are positively related to the expression of MaCSD gene of banana MaCCS gene expression at the transcriptional level and its corresponding the implied resistant process MaCSD gene and MaCCS gene may be involved in the collaboration of banana.

【学位授予单位】：福建农林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S668.1
，

本文编号：1371176