【作者】 李芳军；

【导师】 李召虎； 单立波；

【作者基本信息】 中国农业大学 ， 作物生理， 2014， 博士

【摘要】 棉花(Gossypium spp.)是重要的经济作物之一。生物和非生物胁迫对棉花产量和棉纤维品质造成严重影响,揭示棉花抗逆的分子机制成为研究棉花的重要目标。本研究利用病毒诱导的基因沉默技术(Virus Induce Gene Silencing, VIGS)和转录组测序(RNA-seq)技术,揭示了GhCPS和GhKS参与DPC的调控机制,是DPC作用的靶位点；GhOST1与GhMPK6相互磷酸化正向调控ABA依赖型信号通路；GhBAK1在抗黄萎病机制和细胞死亡中具有正调控作用；成功构建了棉花VIGS cDNA文库。研究结果如下：1.本研究采用同源克隆技术获得了棉花的古巴焦磷酸合酶(Copaly1Pyrophosphate Synthase,CPS)基因GhCPS和内根-贝壳杉合成酶(Ent-Kaurene Synthase,KS)基因GhKS,利用VIGS技术对GhCPS和GhKS的功能进行研究。GhCPS基因全长为2466bp,包含保守的DXDD和SAYDTAW基序与其他物种中CPS的氨基酸序列同源性达50%,GhCPS与PtCPS有较近的亲缘关系；3hKS基因全长为2343bp,包含保守的DDXXD和YDTAWVA基序与其他物种中KS的氨基酸序列同源性达50%,GhKS与CsKS有较近的亲缘关系；DPC处理抑制了GhCPS和GhKS的转录水平；VIGS获得了GhCPS和GhKS功能缺失的棉花突变体植株,其节间伸长受到明显抑制；GA3处理后棉花节间生长恢复,DPC失去抑制作用。2.本研究利用转录组测序技术分析干旱胁迫下棉花的转录组变化。2天干旱处理有102个基因上调表达,4天干旱处理有169个基因上调表达,2天和4天干旱处理同时诱导25个基因上调表达；半定量RT-PCR验证了部分上调基因的转录水平。3.利用VIGS技术鉴定出了两个参与干旱胁迫的基因GhOST1和GhWRKY30. VIGS-GhOST1或VIGS-GhWRKY30的植株对干旱敏感且表型稳定,相对含水量与对照相比显著性下降；VIGS-GhWRKY30的植株对PEG胁迫敏感,对NaCl不敏感；VIGS-GhOST1的植株对PEG和NaCl胁迫均敏感。4.本研究利用体外试验分析GhMPK6与GhOST1两个蛋白激酶之间的互作。免疫共沉淀(Co-Immunoprecipitation, Co-IP)和Pull-Down试验结果证明脱落酸(Abscisic acid, ABA)可诱导GhMPK6与GhOST1的互作；体外激酶活性测定结果表明两者之间能够发生磷酸化转移。5.本研究利用同源比对和VIGS技术分析棉花GhBAK1的基因功能。同源比对分析发现棉花中有2个BAK1的同源基因；VIGS结果表明GhBAK1是棉花抗黄萎病所必须的组分；台盼蓝染色和DAB染色结果显示GhBAK1缺失后棉花叶片细胞死亡,活性氧含量急剧增加。6.本研究成功构建了二倍体棉花的cDNA文库。该文库包括2×106个克隆,库容是棉花基因组的5-10倍；PCR、双酶切鉴定和基因测序结果表明单克隆中插入的片段大小集中在0.5-1Kb之间,且功能各异。更多还原

【Abstract】 Cotton (Gossypium spp.) serves as a economically important crop. However, cotton production is hindered by various biotic and abiotic stresses. The genetic and molecular mechanisms mediating cotton stress responses remain poorly understood. By using RNA-seq and VIGS approach, this studies uncovered that DPC regulated cotton growth via GhCPS and GhKS which were the possible functional sites; GhOST1and GhMPK6could phosphorylate each other to regulate ABA-dependent signaling pathway positively; GhBAK1play positive role in Verticillium wilt resistance and cell death; Cotton VIGS cDNA library was constructed to dissection genes.1. GhCPS and GhKS genes were cloned by homology cloning strategy. The genetic Function of those two was claimed by VIGS. The full length of GhCPS including two conserved domains DXDD and SAYDTAW was2466bp, which had50%amino acid similarity with other species CPS by homology analysis. Phylogenetic analysis indicated GhCPS and PtCPS belonged to the same clade; the full length of GhKS including two conserved domains DDXXD and YDTAWVA was2343bp, which had50%amino acid similarity with other species KS by homology analysis. Phylogenetic analysis indicated GhKS and CsKS belonged to the same clade; DPC treatment could decrease the transcriptional level of GhCPS and GhKS. By loss-of-function analysis, our studies showed that the elongation of cotton internode was inhibited by VIGS-GhCPS or VIGS-GhKS. The inhibition could compromise by GA3treatment.2. This study analysized the transcripton change of cotton upon drought treatment with RNA-seq approach.102and169genes up-regulated by2days or4days drought stress were identified.25genes up-regulated by both2days and4days drought stress; Part of those up-regulated genes were confirmed by semi-quantitative RT-PCR.3. Two genes GhOSTl and GhWRKY30involving in drought tolerance were identified by VIGS. Cotton seedlings of VIGS-GhOSTl or VIGS-GhWRKY30were sensitive to drought. Water lost in cotton leaves of VIGS-GhOST1or VIGS-GhWRKY30were significantly increased; and relative water content of leaves were significantly decreased; cotton VIGS-GhWRKY30were sensitive to PEG but not NaCl; cotton VIGS-GhOST1were sensitive to both treatment.4. This study identified the interaction of two protein kinase GhOST1and GhMPK6by in vitro assay. Co-IP and Pull down assay indicated that ABA could induce the interaction between GhOST1and GhMPK6; In vitro kinase assay indicated that there is a phosphorylation event happened between GhOST1and GhMPK6.5. This study analysized the function of GhBAKl by homology blastn and VIGS. Two BAK1 orthologs were identified in the cotton genome; this study indicated GhBAK1is required for CA4002resistancing to Verticillium wilt by loss-of-function analysis; silencing of GhBAK1is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton.6. A detailed protocol of construction of a VIGS library from diploid cotton G raimondii was established. This library contained2×106clones; sequencing of the representative colonies revealed that the library covers a significant percentage of cotton unique genes; based on the number of colonies in the primary library, it is estimated that this VIGS library is about50×coverage of predicted cotton protein-coding genes; each clone contained diverse gene fragment.更多还原