棉花MKK和MAPK基因家族的全基因组鉴定和功能分析

发布时间：2018-05-30 01:38

  本文选题：MKK + MAPK　； 参考：《南京农业大学》2016年博士论文

【摘要】：棉花是世界上最重要的纤维与油料作物,生物与非生物胁迫严重影响了作物的产量与品质。因此,挖掘参与生物与非生物胁迫相关的抗性基因对提高棉花产量具有重要意义。当植物受到外界胁迫后,会产生各种激素及信号因子。许多研究表明,激素以及逆境可以激活MAPK级联系统,MAPK级联途径在植物对逆境的抗性应答过程中起着至关重要的作用。MAPK级联途径主要由三部分组成,即MAPKKK,MAPKK(MKK)以及MAPK。它们通过连接上游元件和下游受体共同组成一个完整的信号通路。当植物受到外界环境胁迫后,会迅速产生信号分子,激活MAPKKK, MAPKKK通过双重磷酸化激活下游的MKK,再由MKK双重磷酸化激活MAPK,活化的MAPK激酶可能留在细胞质中也可能进入细胞核里,磷酸化下游各种底物,从而将胞外信号传递到胞内调控植物生长发育以及各种胁迫应答。MKK和MAPK基因家族在拟南芥、玉米、水稻、二穗短柄草、苦瓜、番茄、西瓜以及小麦中已经系统研究,但在棉花中尚未有相关系统报道。目前二倍体亚洲棉和雷蒙德氏棉,四倍体陆地棉和海岛棉等4个棉种的基因组测序已完成,为在基因组水平上研究棉花MKK和MAPK基因家族奠定基础。本研究基于棉花现有数据库,通过生物信息学方法预测棉花MKK和MAPK基因家族。根据氨基酸序列和保守基序从雷蒙德氏棉(Gossypium raimondii)中鉴定了 MKK和MAPK家族成员。进一步完成这两个基因家族成员的命名、染色体位置分布、基因结构分类,系统进化、基因复制、表达特点以及两个基因家族成员间蛋白关系分析。基于同源序列比对的方法对棉花现有数据库进行检索,从雷蒙德氏棉中分别鉴定了 11个MKK基因和28个MAPK基因。染色体定位分析表明11个MKK基因定位于13条染色体中的7条染色体上,根据其与拟南芥的同源性命名为GrMKK1-GrMK_2; 28个MAPK基因定位于除D6与D13染色体以外的11条染色体上,根据染色体位置依次命名为GrMPK1-28。MKK和MAPK基因家族成员在染色体上的位置分布均是非冗余的。将MKK与MAPK基因家族成员的氨基酸序列、长度、等电点分子量以及这些基因在细胞中的位置等进行生物信息学分析。序列分析表明MKK基因家族成员编码320-518个氨基酸,亚细胞定位分析表明MKK家族成员主要定位于细胞膜或细胞质。MAPK基因家族成员编码314-658个氨基酸,亚细胞定位分析表明其主要定位于细胞核或细胞质。基于陆地棉((G.hirsutum TM-1)不同组织、器官的转录组信息,以根、茎、叶、花瓣、花药、0DAP胚珠、10DPA纤维、20DPA纤维8个组织器官为材料,研究棉花11个MKK与28个MAPK基因的表达模式。结果发现MKK和MAPK基因在不同组织中均有表达,其中3个MKK与5个MAPK基因在营养器官和生殖器官优势表达;2个MKK与8个MAPK基因在营养器官中高表达;3个MKK与11个MAPK基因在生殖器官中高表达,3个MKK与4个MAPK基因在棉花的各个组织器官都低表达。以陆地棉抗逆品种(晋棉19)三叶期幼苗为实验材料,进行茉莉酸(100μMJA)、脱落酸(1OOμM ABA)、水杨酸(10mM SA)以及H2O2(10mM)等信号分子处理,盐(200mM)、干旱(20%PEG6000)、高温(37℃)、低温(4℃)以及机械损伤等逆境胁迫诱导,通过实时荧光定量PCR技术分析中MKK与MAPK基因家族成员在信号分子及逆境胁迫下的表达模式。结果表明,在信号分子处理的条件下,10个MKK基因至少受一种信号分子诱导上调表达,4个MKK基因受四种信号分子诱导上调表达,1个MKK基因受3信号分子诱导上调表达,3个MKK基因受2种信号分子诱导上调表达,剩下的2个MKK基因只受SA诱导上调表达;23个MAPK基因至少受一种信号分子诱导上调表达,其中10个MAPK基因受四种信号分子诱导上调表达,10个MAPK基因受3中信号分子诱导上调表达,只有1个基因受两种信号分子诱导上调表达,剩下的2个MAPK基因只受JA诱导上调表达。在逆境胁迫处理的条件下,10个MKK基因中MKK7受五种逆境胁迫诱导上调表达,MKK6受四种逆境胁迫诱导上调表达,MKK101和MKK10_2同时受NaCl、4℃和机械损伤诱导上调表达,MKK1和MKK5受两种胁迫诱导上调表达,MKK2_1只受NaCl诱导上调表达,MKK2_2和MKK4则只受机械损伤诱导上调表达,此外,MKK3不受任何逆境诱导上调表达;23个MAPK基因家族成员至少受三种逆境胁迫诱导上调表达,其中8个MAPK基因受五种逆境胁迫诱导上调表达,13个MAPK基因受四种逆境胁迫诱导上调表达,剩余的2个MAPK基因受三种逆境胁迫诱导上调表达。上述结果表明MAPK级联信号途径在信号和逆境胁迫应答过程中起着重要的作用。为阐明MAPK级联途径在植物对逆境胁迫应答过程中的分子机制,根据雷蒙德氏棉中预测的11个MKK基因和28个MAPK基因的序列,利用PrimerPremier5.0软件设计全长引物。以陆地棉TM-1为材料,分别克隆了 8个MKK基因和21个MAPK基因的全长并分别克隆到PGBKT7与PGADT7载体上。将8个MKK分别与21个MAPK成员的重组质粒做共转反应,结果只有63个共转反应可以在四缺(SD/-Ade/-TrP/-His/-Leu)和(SD/-Ade/-Trp/-His/-Leu/ABA/X-α-Gal)正常生长。与拟南芥相比,棉花中的MKK与MAPK家族成员之间的相互作用既有相同的组合也有不同的组合。其中23对MKK-MAPK互作与拟南芥中已报道互作的结果完全一致,40对MKK-MAPK互作是在棉花中的新发现。此外,我们发现8个MKK成员至少与3个MAPK成员互作。反之,一个MAPK也可能与多个MKK之间存在相互作用,如 GhMPK3 和 GhMPK6 都与 GhMKK1、GhMKK2_2、GhMKK5 和 GhMKK6 互作。不同MKK和MAPK之间相互作用,形成多种组合,表明MAPK级联途径在信号转导途径的多样性。比较逆境胁迫处理下棉花中的MKK-MAPK互作模式,发现13条MAPK级联途径可能在棉花对信号分子和逆境的胁迫应答过程中起重要作用。利用反向遗传学技术,研究棉花中MKK与MAPK家族成员在棉花对黄萎病抗性中的功能。利用实时荧光定量PCR技术分析MAPK基因在病原菌诱导处理下的表达特点,结果表明MKK家族成员中MKK1和MKK5和MAPK家族成员中MPK9、MPK13和MPK25受病原菌诱导上调表达。因此,利用VIGS技术进行上述候选基因的抗病功能分析。接种黄萎病菌后,相比于抗性对照材料H7124和注射TRV:00的植株30%的发病率,沉默 MKK1/2_2(65%)、沉默 MKK4/5(83%)、沉默 MPK9 (69.3%)、MPK13(63.75% )和MPK25 ( 54% )的材料都表现了不同程度的发病,且都达到了显著性的差异。这些结果表明MKK基因家族成员中的MKK1/2_2、MKK4/5和MAPK基因家族成员中的MPK9、MPK13、MPK25在棉花对黄萎病抗性应答过程中起重要作用。以上这些结果为棉花中MKK、MAPK家族基因之间的关系及后续功能的研究提供理论基础。
[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.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562
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本文编号：1953353