本文選題：MKK + MAPK��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：棉花是世界上最重要的纖維與油料作物,生物與非生物脅迫嚴重影響了作物的產(chǎn)量與品質(zhì)。因此,挖掘參與生物與非生物脅迫相關(guān)的抗性基因?qū)μ岣呙藁óa(chǎn)量具有重要意義。當植物受到外界脅迫后,會產(chǎn)生各種激素及信號因子。許多研究表明,激素以及逆境可以激活MAPK級聯(lián)系統(tǒng),MAPK級聯(lián)途徑在植物對逆境的抗性應(yīng)答過程中起著至關(guān)重要的作用。MAPK級聯(lián)途徑主要由三部分組成,即MAPKKK,MAPKK(MKK)以及MAPK。它們通過連接上游元件和下游受體共同組成一個完整的信號通路。當植物受到外界環(huán)境脅迫后,會迅速產(chǎn)生信號分子,激活MAPKKK, MAPKKK通過雙重磷酸化激活下游的MKK,再由MKK雙重磷酸化激活MAPK,活化的MAPK激酶可能留在細胞質(zhì)中也可能進入細胞核里,磷酸化下游各種底物,從而將胞外信號傳遞到胞內(nèi)調(diào)控植物生長發(fā)育以及各種脅迫應(yīng)答。MKK和MAPK基因家族在擬南芥、玉米、水稻、二穗短柄草、苦瓜、番茄、西瓜以及小麥中已經(jīng)系統(tǒng)研究,但在棉花中尚未有相關(guān)系統(tǒng)報道。目前二倍體亞洲棉和雷蒙德氏棉,四倍體陸地棉和海島棉等4個棉種的基因組測序已完成,為在基因組水平上研究棉花MKK和MAPK基因家族奠定基礎(chǔ)。本研究基于棉花現(xiàn)有數(shù)據(jù)庫,通過生物信息學(xué)方法預(yù)測棉花MKK和MAPK基因家族。根據(jù)氨基酸序列和保守基序從雷蒙德氏棉(Gossypium raimondii)中鑒定了 MKK和MAPK家族成員。進一步完成這兩個基因家族成員的命名、染色體位置分布、基因結(jié)構(gòu)分類,系統(tǒng)進化、基因復(fù)制、表達特點以及兩個基因家族成員間蛋白關(guān)系分析�；谕葱蛄斜葘Φ姆椒▽γ藁ìF(xiàn)有數(shù)據(jù)庫進行檢索,從雷蒙德氏棉中分別鑒定了 11個MKK基因和28個MAPK基因。染色體定位分析表明11個MKK基因定位于13條染色體中的7條染色體上,根據(jù)其與擬南芥的同源性命名為GrMKK1-GrMK_2; 28個MAPK基因定位于除D6與D13染色體以外的11條染色體上,根據(jù)染色體位置依次命名為GrMPK1-28。MKK和MAPK基因家族成員在染色體上的位置分布均是非冗余的。將MKK與MAPK基因家族成員的氨基酸序列、長度、等電點分子量以及這些基因在細胞中的位置等進行生物信息學(xué)分析。序列分析表明MKK基因家族成員編碼320-518個氨基酸,亞細胞定位分析表明MKK家族成員主要定位于細胞膜或細胞質(zhì)。MAPK基因家族成員編碼314-658個氨基酸,亞細胞定位分析表明其主要定位于細胞核或細胞質(zhì)�；陉懙孛�((G.hirsutum TM-1)不同組織、器官的轉(zhuǎn)錄組信息,以根、莖、葉、花瓣、花藥、0DAP胚珠、10DPA纖維、20DPA纖維8個組織器官為材料,研究棉花11個MKK與28個MAPK基因的表達模式。結(jié)果發(fā)現(xiàn)MKK和MAPK基因在不同組織中均有表達,其中3個MKK與5個MAPK基因在營養(yǎng)器官和生殖器官優(yōu)勢表達;2個MKK與8個MAPK基因在營養(yǎng)器官中高表達;3個MKK與11個MAPK基因在生殖器官中高表達,3個MKK與4個MAPK基因在棉花的各個組織器官都低表達。以陸地棉抗逆品種(晉棉19)三葉期幼苗為實驗材料,進行茉莉酸(100μMJA)、脫落酸(1OOμM ABA)、水楊酸(10mM SA)以及H2O2(10mM)等信號分子處理,鹽(200mM)、干旱(20%PEG6000)、高溫(37℃)、低溫(4℃)以及機械損傷等逆境脅迫誘導(dǎo),通過實時熒光定量PCR技術(shù)分析中MKK與MAPK基因家族成員在信號分子及逆境脅迫下的表達模式。結(jié)果表明,在信號分子處理的條件下,10個MKK基因至少受一種信號分子誘導(dǎo)上調(diào)表達,4個MKK基因受四種信號分子誘導(dǎo)上調(diào)表達,1個MKK基因受3信號分子誘導(dǎo)上調(diào)表達,3個MKK基因受2種信號分子誘導(dǎo)上調(diào)表達,剩下的2個MKK基因只受SA誘導(dǎo)上調(diào)表達;23個MAPK基因至少受一種信號分子誘導(dǎo)上調(diào)表達,其中10個MAPK基因受四種信號分子誘導(dǎo)上調(diào)表達,10個MAPK基因受3中信號分子誘導(dǎo)上調(diào)表達,只有1個基因受兩種信號分子誘導(dǎo)上調(diào)表達,剩下的2個MAPK基因只受JA誘導(dǎo)上調(diào)表達。在逆境脅迫處理的條件下,10個MKK基因中MKK7受五種逆境脅迫誘導(dǎo)上調(diào)表達,MKK6受四種逆境脅迫誘導(dǎo)上調(diào)表達,MKK101和MKK10_2同時受NaCl、4℃和機械損傷誘導(dǎo)上調(diào)表達,MKK1和MKK5受兩種脅迫誘導(dǎo)上調(diào)表達,MKK2_1只受NaCl誘導(dǎo)上調(diào)表達,MKK2_2和MKK4則只受機械損傷誘導(dǎo)上調(diào)表達,此外,MKK3不受任何逆境誘導(dǎo)上調(diào)表達;23個MAPK基因家族成員至少受三種逆境脅迫誘導(dǎo)上調(diào)表達,其中8個MAPK基因受五種逆境脅迫誘導(dǎo)上調(diào)表達,13個MAPK基因受四種逆境脅迫誘導(dǎo)上調(diào)表達,剩余的2個MAPK基因受三種逆境脅迫誘導(dǎo)上調(diào)表達。上述結(jié)果表明MAPK級聯(lián)信號途徑在信號和逆境脅迫應(yīng)答過程中起著重要的作用。為闡明MAPK級聯(lián)途徑在植物對逆境脅迫應(yīng)答過程中的分子機制,根據(jù)雷蒙德氏棉中預(yù)測的11個MKK基因和28個MAPK基因的序列,利用PrimerPremier5.0軟件設(shè)計全長引物。以陸地棉TM-1為材料,分別克隆了 8個MKK基因和21個MAPK基因的全長并分別克隆到PGBKT7與PGADT7載體上。將8個MKK分別與21個MAPK成員的重組質(zhì)粒做共轉(zhuǎn)反應(yīng),結(jié)果只有63個共轉(zhuǎn)反應(yīng)可以在四缺(SD/-Ade/-TrP/-His/-Leu)和(SD/-Ade/-Trp/-His/-Leu/ABA/X-α-Gal)正常生長。與擬南芥相比,棉花中的MKK與MAPK家族成員之間的相互作用既有相同的組合也有不同的組合。其中23對MKK-MAPK互作與擬南芥中已報道互作的結(jié)果完全一致,40對MKK-MAPK互作是在棉花中的新發(fā)現(xiàn)。此外,我們發(fā)現(xiàn)8個MKK成員至少與3個MAPK成員互作。反之,一個MAPK也可能與多個MKK之間存在相互作用,如 GhMPK3 和 GhMPK6 都與 GhMKK1、GhMKK2_2、GhMKK5 和 GhMKK6 互作。不同MKK和MAPK之間相互作用,形成多種組合,表明MAPK級聯(lián)途徑在信號轉(zhuǎn)導(dǎo)途徑的多樣性。比較逆境脅迫處理下棉花中的MKK-MAPK互作模式,發(fā)現(xiàn)13條MAPK級聯(lián)途徑可能在棉花對信號分子和逆境的脅迫應(yīng)答過程中起重要作用。利用反向遺傳學(xué)技術(shù),研究棉花中MKK與MAPK家族成員在棉花對黃萎病抗性中的功能。利用實時熒光定量PCR技術(shù)分析MAPK基因在病原菌誘導(dǎo)處理下的表達特點,結(jié)果表明MKK家族成員中MKK1和MKK5和MAPK家族成員中MPK9、MPK13和MPK25受病原菌誘導(dǎo)上調(diào)表達。因此,利用VIGS技術(shù)進行上述候選基因的抗病功能分析。接種黃萎病菌后,相比于抗性對照材料H7124和注射TRV:00的植株30%的發(fā)病率,沉默 MKK1/2_2(65%)、沉默 MKK4/5(83%)、沉默 MPK9 (69.3%)、MPK13(63.75% )和MPK25 ( 54% )的材料都表現(xiàn)了不同程度的發(fā)病,且都達到了顯著性的差異。這些結(jié)果表明MKK基因家族成員中的MKK1/2_2、MKK4/5和MAPK基因家族成員中的MPK9、MPK13、MPK25在棉花對黃萎病抗性應(yīng)答過程中起重要作用。以上這些結(jié)果為棉花中MKK、MAPK家族基因之間的關(guān)系及后續(xù)功能的研究提供理論基礎(chǔ)。
[Abstract]:Cotton is the most important fiber and oil crop in the world. Biological and abiotic stresses have seriously affected the yield and quality of crops. Therefore, it is of great significance to excavate the resistance genes involved in biological and abiotic stresses to improve the yield of cotton. The study shows that hormone and adversity can activate the MAPK cascade system, and the MAPK cascade plays a vital role in the response of plant resistance to adversity. The cascade pathway of the.MAPK consists mainly of three parts, namely, MAPKKK, MAPKK (MKK) and MAPK., which constitute a complete signal pathway by connecting the upstream elements and downstream receptors. When the plant is stressed by the outside environment, the signal molecules are quickly produced, activating the MAPKKK, activating the MAPKKK through double phosphorylation and activating the downstream MKK, and then activating the MAPK by MKK double phosphorylation. The activated MAPK kinase may remain in the cytoplasm and may enter the nucleus and phosphorylate a variety of downstream substrates to transfer the extracellular signal to the intracellular modulation. Plant growth and stress response.MKK and MAPK gene families have been systematically studied in Arabidopsis, corn, rice, two panicle short stipe, bitter gourd, tomato, watermelon, and wheat, but there are no related system reports in cotton. At present, the diploid Asian cotton and Raymond's cotton, the tetraploid land cotton and the island cotton, and other 4 cotton species Genome sequencing has been completed to lay the foundation for the study of cotton MKK and MAPK gene family at the genomic level. Based on the existing cotton database, this study predicts the MKK and MAPK family of cotton by bioinformatics. The MKK and MAPK families are identified from Raymond's Cotton (Gossypium raimondii) based on the amino acid sequence and the conservative sequence. Members. Further complete the naming of the two gene family members, the distribution of chromosomes, the classification of the gene structure, the phylogenetic evolution, the gene replication, the expression characteristics and the analysis of the protein relationship among the members of the two gene family. Based on the homologous sequence alignment, the existing data base of cotton was retrieved, and 11 of the Raymond S cotton were identified. MKK gene and 28 MAPK genes. Chromosomal location analysis shows that 11 MKK genes are located on 7 chromosomes of 13 chromosomes, and are named GrMKK1-GrMK_2 according to their homology with Arabidopsis; the 28 MAPK genes are located on 11 chromosomes other than D6 and D13 chromosomes, and the root is named GrMPK1-28.MKK and M in sequence according to the location of the chromosomes. The distribution of the APK gene family members on the chromosomes is not redundant. Bioinformatics analysis of the amino acid sequence, length, the molecular weight of the isoelectric point and the location of these genes in the family members of the MKK gene family, and the location of these genes in the cells. Sequence analysis shows that the members of the MKK gene family encode 320-518 amino acids and subcellular localization points. The analysis showed that the members of the MKK family mainly located in the cell membrane or cytoplasmic.MAPK gene family members encoding 314-658 amino acids. The subcellular localization analysis showed that it was mainly located in the nucleus or cytoplasm. Based on the different tissues of G.hirsutum TM-1, the transcriptional group of the organs, the roots, stems, leaves, petals, anthers, 0DAP ovules, 10DPA fibers, 8 tissues and organs of 20DPA fiber were used to study the expression patterns of 11 MKK and 28 MAPK genes in cotton. The results showed that MKK and MAPK genes were expressed in different tissues, of which 3 MKK and 5 MAPK genes were expressed in the vegetative organs and reproductive organs; 2 MKK and 8 MAPK genes were highly expressed in the vegetative organs; 3 MKK and 11 MAPK bases were expressed. Because of high expression in the reproductive organs, 3 MKK and 4 MAPK genes are low expressed in every tissue and organ of cotton. The experimental materials of three leaf stage seedlings of Upland Cotton (Jin cotton 19), jasmonic acid (100 mu MJA), abscisic acid (1OO mu M ABA), salicylic acid (10mM SA) and H2O2 (10mM), and other signal molecules, salt (200mM), drought (20%PEG6000), are used as experimental materials. High temperature (37 C), low temperature (4) and mechanical damage were induced, and the expression patterns of MKK and MAPK gene family members under signal molecules and stress were analyzed by real time fluorescence quantitative PCR. The results showed that, under the condition of signal molecule treatment, 10 MKK genes were induced up to up expression by one kind of signal molecules, 4 MKK The gene was induced up up expression by four signal molecules, 1 MKK genes were induced up regulation by 3 signal molecules, 3 MKK genes were induced up to up expression by 2 signal molecules, and the remaining 2 MKK genes were only up regulated by SA, and 23 MAPK genes were induced by at least one kind of signal molecules, of which 10 MAPK genes were subjected to four signal molecules. Induced up regulation, 10 MAPK genes are up regulated by 3 signal molecules, only 1 genes are regulated by two signal molecules, and the remaining 2 MAPK genes are only up regulated by JA. Under the condition of stress treatment, 10 MKK genes are regulated by five kinds of inverse boundary stress, and MKK6 is subjected to four stress stresses. The expression of MKK101 and MKK10_2 is up regulated by NaCl, 4 C and mechanical damage, MKK1 and MKK5 are up regulated by two stresses, MKK2_1 is only up regulated by NaCl, MKK2_2 and MKK4 are only up regulated by mechanical damage induction, and MKK3 is not up regulated by any adversity induction; 23 MAPK gene family members Three kinds of stress induced up-regulated expression were induced by three stresses, 8 of which were induced up regulated by five stresses, 13 MAPK genes were induced by four stress stress, and the remaining 2 MAPK genes were regulated by three stresses. The results showed that the MAPK cascade signal pathway was in the signal and stress response process. In order to clarify the molecular mechanism of the MAPK cascade pathway in response to stress response in plants, the full length primers were designed by PrimerPremier5.0 software based on the sequence of 11 MKK and 28 MAPK genes predicted in Raymond S's cotton. 8 MKK genes and 21 MAPK groups were cloned from TM-1 as the material of land cotton. The total length of the cause was cloned to the PGBKT7 and PGADT7 vectors. The recombinant plasmids of 8 MKK and 21 MAPK members were co rotating, and only 63 co rotation reactions could grow normally in four (SD/-Ade/-TrP/-His/-Leu) and (SD/-Ade/-Trp/-His/-Leu/ABA/X- alpha -Gal). Compared with Arabidopsis, the MKK and the MAPK family members in cotton There are both the same combinations and different combinations. 23 pairs of MKK-MAPK interactions are fully consistent with the results reported in Arabidopsis, and 40 pairs of MKK-MAPK interactions are new discoveries in cotton. In addition, we found that 8 MKK members are at least 3 MAPK members. One MAPK may also exist with multiple MKK. Interaction, such as GhMPK3 and GhMPK6, interacts with GhMKK1, GhMKK2_2, GhMKK5 and GhMKK6. The interaction between different MKK and MAPK forms a variety of combinations, indicating the diversity of the signal transduction pathway in the MAPK cascade pathway. Compare the interaction patterns of MKK-MAPK in cotton under stress stress, and find that 13 MAPK cascade pathways may be in cotton. The function of MKK and MAPK family members in cotton resistance to Verticillium wilt was studied by using reverse genetics technology. The expression of MAPK gene was analyzed by real time fluorescence quantitative PCR. The results showed that MKK1 and MKK5 in MKK family members were MKK1 and MKK5. MPK9, MPK13 and MPK25 in the MAPK family were up-regulated by pathogenic bacteria. Therefore, VIGS technology was used to analyze the disease resistance of the candidate genes. After inoculation of Verticillium wilt, compared to the incidence of resistance control material H7124 and the incidence of plant 30% injected with TRV:00, MKK1/ 2_2 (65%), silencing MKK4/5 (83%), silence MPK9 (69.3%), MPK13) The (63.75%) and MPK25 (54%) materials all showed different degrees of morbidity and reached significant differences. These results showed that the MPK9, MPK13, MPK25 in the members of the MKK gene family, MPK13, and MPK25 played an important role in the response process of cotton to Verticillium wilt resistance. The above results are MK in cotton. K provides a theoretical basis for the study of the relationship between MAPK family genes and subsequent functions.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S562
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本文編號：1953353