【作者】 李宁；

【导师】 高杰；

【作者基本信息】 新疆农业大学 ， 园艺学， 2021， 博士

【摘要】 盐胁迫影响番茄的整个生长发育进程,种子萌发和幼苗期的番茄对盐分较为敏感。野生番茄潘那利(Solanum pennellii)耐盐性状优异,是番茄抗逆性育种不可多得的研究材料。本研究通过下一代测序技术,首次鉴定了栽培番茄M82和野生番茄潘那利中盐胁迫响应相关的编码及非编码RNA,同时基于生物信息学手段构建了m RNA和micro RNA、lnc RNA之间的竞争性表达失调网络,筛选了耐盐番茄潘那利中盐胁迫核心响应基因元件及信号通路,并针对核心基因进行原核表达、亚细胞定位和转基因体系构建等功能组学研究,揭示了野生番茄潘那利盐胁迫响应机理。为番茄培育耐盐品种及耐盐分子机制提供理论依据,为农作物抗逆育种提供候选基因资源,同时对促进番茄生态产业多元化具有重要应用价值。(1)以盐胁迫处理0 h和12 h的潘那利番茄与栽培番茄M82的根为材料,进行全转录组测序分析。分析在盐胁迫12 h下两个品种中lnc RNAs表达模式和潜在靶基因的差异。总共鉴定了1044个假定的lnc RNAs,它们分布在所有染色体上。其中1号染色体上的lnc RNAs最多,有117个,2号和6号染色体上只有58个。在盐胁迫下137个lnc RNAs在野生番茄中差异表达,其中37个上调,100个下调。GO和KEGG分析显示,125个DE-lnc RNAs靶向M82中的1227个靶基因,而111个DE-lnc RNAs靶向潘那利番茄中的1268个m RNAs。确定9个lnc RNAs为10个已知的番茄mi RNA前体,包括mi R319a、mi R396b、mi R7981c/d/e/f、mi R10532、mi R10538、mi R1919b和mi R1919c。M82和潘那利番茄中分别有23和21个lnc RNAs分别被判定为34和28个mi RNAs的可能靶点,其中与非生物胁迫有关的mi RNA,有sly-mi RNA159、slymi RNA319A等。(2)对栽培番茄M82和潘那利番茄mi RNA-seq数据分析,所有样品结果共得到370个mi RNAs,其中64个为已知mi RNAs,306个为新预测的mi RNA。新预测的mi RNA中有213个长度为24 nt,占比69.61%,新预测的24 nt的mi RNAs中,潘那利番茄数目要远高于栽培番茄,该长度mi RNA主要来源于基因组重复序列。盐胁迫处理下M82和潘那利番茄之间存在27个差异表达mi RNAs,其中5个mi RNAs为上调表达,22个mi RNAs为下调表达,16个mi RNAs为已知mi RNA,且全部为下调表达,其中12个mi RNAs为新鉴定mi RNA。盐胁迫下两种番茄所有差异表达mi RNA共预测到681个靶基因,其中15个已知差异表达mi RNAs靶向341个靶基因,10个新预测mi RNAs靶向340个靶基因。差异表达mi RNAs靶基因的注释结果显示,差异靶基因大量分布在糖酸代谢通路中,并被特异富集在丙氨酸、天冬氨酸和谷氨酸代谢、甘氨酸、丝氨酸和苏氨酸代谢、赖氨酸降解等7个氨基酸代谢通路中。差异表达mi RNA及其靶向的靶基因联合分析,最终获得13个已知mi RNAs调控15个m RNAs,其中slymi R482b、sly-mi R164a-5p、sly-mi R166c-3p和sly-mi R9470-3p分别调控NBS-LRR、NAC、HD-ZIP和ERF等转录因子家族成员,sly-mi R156d-3p和sly-mi R395a靶向调控ACAD10和硫酸盐转运体的表达。(3)本研究通过组学数据综合分析,筛选确定CDPK为研究对象。基于CDPK基因家族保守结构域,利用HMMER软件和本地Blastp方法对潘那利番茄基因组进行检索,共鉴定到31个Sp CDPKs;系统发育分析显示所有Sp CDPK基因分为4个亚家族;潘那利番茄Sp CDPK家族基因数目少于拟南芥中的34个,但多于栽培番茄中的29个;基因结构和剪切位点显示,Sp CDPK家族基因存在较为复杂的外显子-内含子模式和大量m RNA异构体表达潜力;物种间共线性表明潘那利番茄与M82中的CDPK基因保持着高度同源,他们可能参与介导相同或相似功能;Sp CDPK扩增机理的研究发现全基因组复制倍增是潘那利番茄CDPK家族基因扩张的主要原因;相互作用蛋白结果显示Sp CDPK与大量离子运输蛋白协同表达,参与潘那利番茄多种生命活动;最后基于RNA-seq的技术对潘那利番茄中所有Sp CDPKs盐胁迫响应机制进行了研究,依据高盐环境下Sp CDPKs差异表达情况,初步筛选了对潘那利番茄耐盐能力提升具有积极意义的7个Sp CDPKs作为候选基因,为下一步基因可能和功能解析提供了基础。(4)基于同源克隆技术,从野生潘那利番茄中获得Sp CDPK4的全长序列,Sp CDPK4的开放阅读框长度为1746 bp,编码581个氨基酸,理论蛋白分子量约为64.58 k Da,等电点为5.54,是非分泌型、亲水的稳定蛋白,且定位于细胞膜上;多序列比对及系统发育树分析表明,Sp CDPK4编码的氨基酸序列与栽培番茄Sl CDPK4(XP＿010327694.1)、马铃薯St CDPK4(NM＿001287877)相似度最高,相似性均为65.1%,与拟南芥At CDPK20亲缘关系最近归为第二类。SDS-PAGE电泳检测结果发现在70 k Da左右处有一条特异表达的蛋白条带,与预期目的产物大小一致,且WesternBlot结果显示其能与Anti-His单克隆抗体发生特异性反应。过表达Sp CPK4能显著增强重组大肠杆菌对盐的耐受性。RT-q PCR结果表明,Sp CDPK4在潘那利番茄不同组织中均有表达,且根中的表达量显著高于叶和茎;此外在盐胁迫下,Sp CDPK4在根中的表达量迅速积累,且在1 h时达到峰值。转基因番茄盐胁迫处理48 h后,对照植株明显萎蔫严重,初步说明该基因可能与植物的耐盐性相关。更多还原

【Abstract】 Tomato is under constant threat of salt stress throughout its reproductive life,its seed germination and seedling stages are sensitive to salt.Wild tomatoes have excellent stress tolerance traits.Solanum pennellii has a high tolerance to salt stress and is a very important research material for tomato stress tolerance breeding.In this study,we first identified coding and non-coding RNAs related to salt stress response in cultivated tomato M82 and S.pennellii by next-generation sequencing,and constructed a competitive expression dysregulation network among m RNA,micro RNA and lnc RNA based on bioinformatics,and screened the core gene components and signaling pathways for salt stress response in S.pennellii.We have also developed functional genomic studies on the core genes,including prokaryotic expression,subcellular localization and transgenic system construction,to reveal the mechanism of salt stress response in S.pennellii.It provides a theoretical basis for the cultivation of new salt-tolerant varieties and molecular mechanisms of salt tolerance in tomato,provides candidate gene resources for crop resistance breeding,and also has important application value for promoting diversified production of tomato eco-industry.(1)Whole transcriptome sequencing analysis was performed on the roots of wild tomato S.pennellii and cultivated tomato M82 treated with salt stress for 12 h.The lnc RNAs identified in the two cultivars were compared,and the differences in the expression patterns of lnc RNAs and potential target genes in the two cultivars under salt stress for 12 h were analyzed.A total of 1044 putative lnc RNAs were identified,which were distributed on all chromosomes.The largest number of lnc RNAs was found on chromosome 1 with 117 and only 58 on chromosomes 2 and 6.Expression levels of lnc RNAs under salt stress were analyzed,137 lnc RNAs were differentially expressed in wild tomato,of which 37 were upregulated and 100 were down-regulated.GO and KEGG analyses showed that 125 DElnc RNAs targeted 1227 target genes in M82,while 111 DE-lnc RNAs targeted 1268 m RNAs in S.pennellii.Nine lnc RNAs were identified as 10 known tomato mi RNA precursors,including mi R319 a,mi R396 b,mi R7981c/d/e/f,mi R10532,mi R10538,mi R1919 b,and mi R1919 c.23 and 21 lnc RNAs were identified as possible targets for 34 and 28 mi RNAs in M82 and Pannari,respectively.in which related to abiotic stress,including sly-mi RNA159 and sly-mi RNA319 A.(2)A total of 370 mi RNAs were obtained by mi RNA-seq analysis of M82 and S.pennellii,of which 64 were known mi RNAs and 306 were newly predicted mi RNAs.213 of the newly predicted mi RNAs were 24-nt in length,accounting for 69.61%.The number of S.pennellii newly predicted 24-nt mi RNAs was much higher than that in cultivated tomatoes,and mi RNAs of this length were mainly derived from genomic repeat sequences.There were27 differentially expressed mi RNAs between M82 and S.pennellii tomatoes under salt stress treatment,of which 5 mi RNAs were up-regulated,22 mi RNAs were down-regulated,16 mi RNAs were known mi RNAs and all of them were down-regulated,and 12 mi RNAs were newly identified mi RNAs.A total of 681 target genes were predicted for all differentially expressed mi RNAs,of which 15 known differentially expressed mi RNAs targeted 341 target genes and 10 newly predicted mi RNAs targeted 340 target genes.Annotation of differentially expressed mi RNA target genes between M82 and S.pennellii showed that differential target genes were also abundantly distributed in the glycolate metabolic pathway,and specifically enriched in seven amino acid metabolic pathways,including alanine,aspartate and glutamate metabolism,glycine,serine and threonine metabolism,and lysine degradation.The combined analysis of M82 and S.pennellii differentially expressed mi RNAs and their target genes after salt stress resulted in 13 known mi RNAs regulating 15 m RNAs,of which sly-mi R482 b,slymi R164a-5p,sly-mi R166c-3p and sly-mi R9470-3p regulate transcription factor family members,such as NBS-LRR,NAC,HD-ZIP and ERF,respectively,and sly-mi R156d-3p and sly-mi R395 a target and regulate the expression of ACAD10 and Sulfate transporter.(3)In this study,CDPK was selected as the research object through the comprehensive analysis of the previous omics data.Based on the conserved structural domains of the CDPK gene family,HMMER software and the native Blastp method were used to identify 31 Sp CDPKs from the whole S.pennellii genome and named them according to their position on the S.pennellii chromosome;phylogenetic analysis showed that all Sp CDPKs genes were divided into five subfamilies,and the number of Sp CDPK genes in S.pennellii was less than34 in Arabidopsis but more than 29 in cultivated tomato;gene structure and shearing sites showed that Sp CDPK has a complex exon-intron pattern and a large number of m RNA heterodimeric expression potential,and interspecies covariation indicated that the CDPK genes in S.pennellii and M82 remain highly homologous and they may be involved in mediating the same or similar functions,The study of Sp CDPK amplification mechanism revealed that genome-wide replication ploidy was the main reason for the expansion of CDPK family genes in S.pennellii,and the results of interacting proteins showed that Sp CDPK was co-expressed with a large number of ion transport proteins and involved in various life activities in S.pennellii.Based on the differential expression of Sp CDPKs in high-salt environments,seven Sp CDPKs with positive effects on salt tolerance in S.pennellii were initially screened as candidate genes,providing a basis for the next step of possible gene and function analysis.(4)Based on homologous cloning technique,the full-length sequence of Sp CDPK4 was obtained from S.pennellii.Sp CDPK4 has an open reading frame length of 1746 bp,encodes581 amino acids,has a theoretical protein molecular weight of approximately 64.58 k Da,and an isoelectric point of 5.54.It is a non-secretory,hydrophilic,stable protein and was localized at cell membrane.Multiple sequence alignment and phylogenetic tree analysis showed that the amino acid sequence encoded by Sp CDPK4 had the highest similarity with cultivated tomato Sl CDPK4(XP＿010327694.1)and potato St CDPK4(NM＿001287877),both with65.1% similarity,and the closest affinity to Arabidopsis At CDPK20 was classified as the second class.SDS-PAGE electrophoresis revealed a specifically expressed protein band at around 70 k Da,consistent with the expected size of the target product,and Western-Blot results showed that it could react specifically with Anti-His monoclonal antibody.Overexpression of Sp CPK4 could significantly enhance the salt tolerance of recombinant Escherichia coli.RT-q PCR results showed that Sp CDPK4 was expressed in different tissues of S.pennellii,and the expression of Sp CDPK4 was significantly higher in roots than in leaves and stems.In addition,the expression of Sp CDPK4 in roots accumulated rapidly under salt stress and reached a peak at 1 h.After 48 h of salt stress treatment in transgenic tomato,the control plants wilted significantly and severely,tentatively suggesting that this gene may be associated with salt tolerance of plants.更多还原