發(fā)布時間：2018-01-02 22:22

  本文關鍵詞：香蕉SOD基因家族的全基因組鑒定及功能分析　出處：《福建農(nóng)林大學》2016年博士論文　論文類型：學位論文

  更多相關文章： 香蕉 SOD基因家族 啟動子 非生物脅迫 激素處理 表達分析

【摘要】：香蕉是熱帶和亞熱帶地區(qū)重要的經(jīng)濟和糧食作物。但在生產(chǎn)上,易受冬春季低溫和夏季干旱等不利環(huán)境的影響,而導致植株生長發(fā)育受阻或減產(chǎn),給蕉農(nóng)造成較大經(jīng)濟損失。逆境脅迫往往誘導植物細胞內(nèi)活性氧自由基的過量積累,進而產(chǎn)生氧化脅迫,影響植物的生長發(fā)育和結(jié)果。超氧化物歧化酶(Superoxide dismutase,SOD)是抗氧化系統(tǒng)的第一道防線,能有效的清除活性氧并減輕氧化脅迫從而提高植物對逆境的耐受能力。已有研究表明香蕉SOD同工酶譜和酶活性在低溫等非生物脅迫下發(fā)生顯著改變,說明SOD與香蕉抗逆境過程密切相關。但目前對香蕉SOD基因及其表達的調(diào)控機制還知之甚少。因此,本研究以兩個野生蕉的全基因組序列為參考,在栽培香蕉中對SOD基因家族進行系統(tǒng)的克隆鑒定,分析不同成員的啟動子序列和順式調(diào)控元件,對不同SOD基因成員在非生物脅迫和激素處理下的表達調(diào)控進行研究,以探討SOD在香蕉抗逆過程中的作用。主要研究結(jié)果如下：1.香蕉SO1)基因家族的全基因組分析與克�、俨捎糜嬎銠C分析方法從小果野蕉DH-Pahang(Musa acuminata,AA genome)的全基因組中共檢索到15條SOD候選序列,其中2條為冗余序列。從野蕉PKW(Musa balbisiana,BB genome)的全基因組中共檢索到14條SOD候選序列,其中1條為冗余序列,2條為嵌合基因。②根據(jù)兩個野生蕉的SOD序列設計引物,以福建天寶蕉(Cavendish banana,AAA genome)為材料,共克隆得到25條不同的SOD mRNA轉(zhuǎn)錄本。它們分別由6個Cu/ZnSOD基因、4個MnSOD基因和2個FeSOD基因轉(zhuǎn)錄�？勺兗艚�、可變轉(zhuǎn)錄起始位點和3'UTR選擇性多聚腺苷酸化是導致MaSOD基因mRNA多樣性的原因。③以DNA為模板,克隆了12個MaSOD家族基因的gDNA序列,大小為1807～4720 bp。根據(jù)基因結(jié)構(gòu)不同分為4組：MaCSD1A、MaCSD1B、MaCSD1C和MaCSD1L共4個成員為Ia組,均含有6個內(nèi)含子。MaCSD2A和MaCSD2B構(gòu)成Ib組,均含有7個內(nèi)含子。4個MnSOD (MaMSD1A、MaMSD1B、MaMSD1C和MaMSD1D)組成II組,均含有5個內(nèi)含子。III組包含MaFSD1A和MaFSD11共2個成員,但它們的外顯子數(shù)目存在差異；MaFSD1A含有7個內(nèi)含子,而MaFSD1B含有8個內(nèi)含子。該分類結(jié)果與MaSOD家族基因氨基酸序列的聚類結(jié)果及根據(jù)它們保守基序的分類結(jié)果一致。④物種內(nèi)和物種間的共線性分析結(jié)果表明,全基因組復制和染色體區(qū)段復制是香蕉SOD基因家族數(shù)量擴張的主要因素。⑤對不同基因組類型香蕉SOD家族基因的比較分析結(jié)果表明,福建天寶蕉(AAA genome)的SOD基因與福州野生蕉(AA genome)和小果野蕉DH-Pahang(AA genome)的SOD基因有較高的一致性,但與野蕉PKW (BB genome)的SOD基因的一致性較低。2.香蕉SOD基因家族編碼蛋白的生物信息學分析根據(jù)生物信息學分析結(jié)果,香蕉SOD家族基因編碼的蛋白與其他植物SOD具有很高的同源性,且都含有SOD保守結(jié)構(gòu)域和特征氨基酸位點,說明他們在進化上較為保守,在功能上具有相似性。但不同MaSOD蛋白仍存在各自的特點。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為堿性蛋白,MaCSD2B 和 MaMSDIA為弱堿性蛋白外,其余的成員均為酸性蛋白。除了MaFSD1A為不穩(wěn)定蛋白外,其余的11個成員均為穩(wěn)定蛋白。除了MaCSD2A 和 MaCSD2B為疏水性蛋白外,其余的均為親水性蛋白。MaCSD1的4個蛋白(MaCSD1A、MaCSDIB、MaCSD1C 和 MaCSD1D)由19種氨基酸組成,都不含有色氨酸；其余的MaSOD家族蛋白均由20種氨基酸組成。亞細胞定位分析顯示4個MaCSD1蛋白(MaCSD1A、MaCSD1B、MaCSD1C和MaCSD1D)定位于細胞質(zhì)；2個MaCSD2蛋白(MaCSD2A 和 MaCSD2B)定位于葉綠體；4個MaMSD蛋白(MaMSD1A、MaMSD1B、 MaMSD1C 和 MaMSD1D)定位于線粒體；而MaFSD1A主要定位于葉綠體,在細胞質(zhì)中也存在,MaFSD1B則主要定位于葉綠體。磷酸化位點預測分析結(jié)果表明不同MaSOD蛋白成員間的各種磷酸化位點的數(shù)量和位置存在明顯差異,說明香蕉MaSOD家族的不同蛋白成員可能在翻譯后水平受不同的磷酸化方式調(diào)控表達。3.香蕉SOD基因家族啟動子的克隆與分析采用PCR法直接克隆得到MaSOD家族的11條大小在1084～2114 bp的5’端調(diào)控序列。序列分析顯示,它們不僅包含核心啟動子區(qū)和啟動子核心元件TATA-box 和 CAAT-box,還含有大量與光響應、環(huán)境脅迫應答、激素響應和生理節(jié)律調(diào)控等相關的順式元件。對MaSOD基因家族不同成員的啟動子順式元件的比較分析表明,不同成員的啟動子間除了都含有各自特異的順式元件外,還具有一些共有的順式元件以及響應同一脅迫的不同順式元件。說明在一定程度上,同一脅迫可以調(diào)控多個香蕉SOD成員的表達,但各個成員在響應同一脅迫上又具有一定的差異性。4.香蕉SOD基因家族在非生物脅迫和激素處理下的表達分析qRT-PCR法分析MaSOD基因家族在不同組織部位的表達情況,結(jié)果表明除了MaCSD2B基因在假莖中未檢測到表達外,其余的11個成員在葉、假莖和根中都有表達。qRT-PCR法分析MaSOD基因家族在低溫、高溫、干旱和NaCL脅迫下的表達模式。結(jié)果表明香蕉MaSOD基因在不同非生物脅迫下,并非是簡單的一對一響應,而是存在較為復雜的綜合響應。具體情況如下：在低溫脅迫48 h時,香蕉MaCSD2A基因顯著上調(diào)表達,而MaCSD1D和MaCSD2B則顯著下調(diào)表達,其余成員的表達差異不明顯。在高溫脅迫48 h時,有6個基因(MaCSD1B、MaCSD1D、MaMSD1A、MaMSD1B、MaMsD1C和MaFSD1A)呈現(xiàn)顯著上調(diào)表達,只有MaCSD2A基因在12h時顯著下調(diào)表達。而在干旱脅迫下,只有3個Cu/ZnSOD基因(MaCSD1B、MaCSD1C和MaCSD2A)在不同的時間點顯著上調(diào)表達；其余的成員多數(shù)表達量呈現(xiàn)下調(diào),其中5個成員(MaCSD1A、MaCSD1D、MaMSD1A、 MaMSD1B和MaFSD1B)表達量的下調(diào)倍數(shù)達2～10倍。高鹽脅迫顯著誘導MaCSD1D、 MaMSDIA和MaMSD1B基因的表達,但抑制MaCSD2A和MaFSD1B基因的表達。qRT-PC R法分析MaSOD基因家族在脫落酸、赤霉素、生長素和水楊酸4種激素處理下的表達模式。結(jié)果表明只有MaCSD和MaMSD亞家族的成員被顯著誘導表達,而MaFSD亞家族的成員表達量變化不明顯。具體情況如下：在脫落酸處理下,MaCSD1D和MaMSD1A被誘導顯著上調(diào)表達。在赤霉素處理后,也僅有2個基因(MaCSD1D和MaMSD1A)在不同時間點顯著上調(diào)表達,其他成員的表達差異不顯著。生長素處理則可誘導3個MaCSD基因(MaCSDIA、MaCSD1D和MaCSD2B)和1個MaMSD基因(MaMSD1A)在不同時間點顯著上調(diào)表達。而水楊酸處理24 h時顯著誘導MaCSD1D的上調(diào)表達。5.香蕉銅鋅超氧化物歧化酶MaCSD1D基因的功能分析細胞質(zhì)型的MaCSD1D基因在非生物脅迫(冷、熱、干旱和鹽脅迫)和激素處理(ABA、GA3、IAA和SA)下均有顯著的上調(diào)或下調(diào)表達,暗示該基因可能在香蕉的逆境脅迫中發(fā)揮重要作用。為了進一步揭示其功能,構(gòu)建MaCSD1D基因的過表達載體并轉(zhuǎn)化煙草,對MaCSD1D基因在低溫脅迫下的功能進行分析,結(jié)果表明轉(zhuǎn)MaCSD1D基因的煙草種子在低溫下的萌發(fā)速度和生長狀況均優(yōu)于非轉(zhuǎn)基因煙草的種子。6片功能葉時期的轉(zhuǎn)基因煙草比非轉(zhuǎn)基因煙草對低溫脅迫表現(xiàn)出更強的耐受能力,且轉(zhuǎn)基因煙草在低溫下的SOD酶活性高于非轉(zhuǎn)基因煙草的SOD酶活性。6.香蕉Cu/ZnSOD分子伴侶蛋白基因MaCCS的克隆與表達分析銅鋅超氧化物歧化酶(Cu/ZnSOD)是一種同時含銅和鋅的金屬酶,在正常生理條件下銅的獲取需要通過分子伴侶蛋白CCS來實現(xiàn)。本研究采用RT-PCR結(jié)合RACE-PCR獲得了1條新的CCS基因(MaCCS)。序列分析表明MaCCS具有典型的CCS結(jié)構(gòu)域和保守的基因結(jié)構(gòu)。MaCCS基因啟動子上存在大量參與非生物脅迫和激素應答的順式元件。qRT-PCR分析表明MaCCS基因在葉、假莖和根部均有表達,并參與非生物脅迫(CuS04、熱、冷和干旱)和激素(ABA和IAA)應答。而且,MaCCS基因的轉(zhuǎn)錄模式在低溫脅迫下與MaCSD1B、 MaCSD1D和MaCSD2B基因的相似,在熱脅迫下與MaCSD1B和MaCSD1D基因的相似,在干旱脅迫下與MaCSD1B和MaCSD1C基因的相似,在ABA和IAA處理下則與MaCSD1A、 MaCSD1C和MaCSD2B基因的相似。這說明在非生物脅迫和激素處理下,香蕉MaCCS基因在轉(zhuǎn)錄水平的表達與其對應的MaCSD基因的表達呈現(xiàn)正相關,暗示MaCCS基因可能與MaCSD基因協(xié)作參與香蕉的抗逆過程。
[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.

【學位授予單位】：福建農(nóng)林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S668.1
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本文編號：1371176