【作者】 杨作仁；

【导师】 李付广；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2014， 博士

【摘要】 棉花是世界上最重要的纤维作物。世界人口迅速增长，可用耕地持续减少，只有提高亩产量和纤维品质才能应对全球持续增长的棉花需求。陆地棉(Gossypium hirsutum L.)是异源四倍体棉花，也是种植面积最广的棉花，大概占到全部棉花产量的95%。株高不仅是影响棉花株型的决定因素，也是决定产量的重要农艺性状。半矮化且侧枝较短的株型能够使棉花充分利用空间结构，增强抗倒伏能力，获得最佳产量。油菜素内酯（BR）是调节植物株型的重要因素，大田喷施和体外培养也已证实油菜素内酯能够促进棉花纤维的伸长，但是BR调节纤维伸长的机制仍不清楚。本研究以陆地棉T-DNA插入矮化突变体pag1(pagoda1)为研究材料，研究了其形态特征、生理生化特点和遗传特性，利用激活标签克隆了造成突变表型的基因，验证了基因功能；利用该油菜素内酯缺陷型突变体探究BR影响纤维发育的分子机理，为棉花株型的改良、产量和纤维品质的提高提供理论依据和支撑。主要研究结果如下：1.首次利用T-DNA激活标签技术，从四倍体棉花中创制了功能获得型突变体pag1， pag1表现出叶片褶皱暗绿、叶柄缩短、育性降低、纤维缩短和强度降低。细胞学观察表明pag1矮化紧缩的表型是由于细胞的伸长和扩展受到抑制造成的。在黑暗条件下pag1仍具有光形态建成反应，施加外源油菜素内酯能够恢复其生长，并且pag1中BR合成的基因受到反馈调节而表达上调，表明pag1不是一个BR不敏感型突变体而是BR缺陷型突变体。2.遗传分析表明该突变体是由显性单基因控制的，并且突变表型和T-DNA标签紧密连锁，进一步分析表明激活标签插入到一个P450基因的上游，表达分析证实该基因被激活表达。在拟南芥中超表达该基因后能够重演pag1的矮化表型，并且矮化表型可以被BL处理所恢复。上述结果表明该基因的超表达就是造成pag1矮化的原因，将其命名为PAG1。PAG1的表达受BR和光诱导，PAG1与CYP734A1相似程度较高，拟南芥CYP734A1能够使体内活性BR的失活，因此PAG1可能也参与了类似的BR失活反应。3.利用组成型启动子驱动PAG1可获得基因表达量不同的转化体，且转化体的株高与基因表达量成反比，该结果表明可以通过调控PAG1的表达量来调节转基因植物的株高。将来自于棉花的PAG1基因转化单子叶模式植物水稻后也能造成矮化的表型，这表明PAG1也可调节单子叶植物的株型。BR局部处理和嫁接实验证明BR在棉花体内不能进行长距离运输，这为利用PAG1特异调节内源BR含量提供了可能。利用绿色组织特异启动子驱动PAG1转化拟南芥后能够获得对生殖生长影响较弱但株型得到明显改变的转化体，这表明可以利用组织特异的启动子驱动PAG1的表达特定地改良植物株型。4. BR的合成和代谢同时进行，精密调控内源BR含量，以保证植物的正常生长和发育。PAG1在棉花纤维的起始和伸长期表达提高，可能在棉花纤维发育中起重要作用。pag1的成熟纤维长度显著缩短；体外胚珠培养条件下，pag1纤维仍然显著短于野生型，而在培养基中添加BL后能够恢复其纤维伸长，表明PAG1过表达导致内源BR失活从而抑制了纤维伸长。纤维的转录组分析表明，PAG1通过调节内源BR含量影响纤维发育，BR可能是通过影响超长链脂肪酸的合成来调节乙烯信号转导途径；BR缺陷影响了脂肪酸合成、乙烯信号转导、钙离子信号转导、细胞壁、细胞骨架和细胞生长相关基因的表达；这暗示BR可能是纤维发育的中枢调节因子，其处于调节纤维发育相关因子的上游，通过影响其他纤维发育相关途径调控纤维伸长。通过特异调节PAG1的表达有望在棉花株型和纤维品质改良方面发挥重要作用。更多还原

【Abstract】 Cotton is the most important textile crop in the world. The world’s population is growing fast,however, the available land continues to decline. Improving the acre yield and fiber quality is the onlyway to satisfy the world demands. Upland cotton (Gossypium hirsutum), an allotetraploid variety, is themost widely cultivated cotton species, accounting for95%of production worldwide. Plant height is notonly a determinant factor of plant architecture, but also an essential agronomic trait determining yield.An ideal cotton architecture includes semi-dwarf and shorter lateral branches, which allows a cottonplant to most efficiently use its three dimensional space and to produce the highest yield.Brassinosteriods (BR) is one of the most important factors effecting plant architecture. Sparing in thecotton field and invitro culture of ovules have proved that BR promote fiber elongation, nevertheless,the mechanism of BR regulation of fiber elongation remains elusive. In this paper, we reported ouridentification of a cotton activation-tagged mutant, and described its morphological, genetic,physiological and biochemical characteristics. The gene that caused the dwarf phenotype was clonedusing the T-DNA tag and verifed by transgenic research. The molecular mechanism of BR regulation offiber elongation was explored using the BR-deficiency mutant.1. We isolated the first gain of function mutant of allotetraploid cotton using theactivation-tagging technique. pag1shows extreme dwarfism, wrinkled and dark green leaves,shortened petioles, decreased pollen activity. Histological analysis revealed that cellelongation and expansion were largely reduced in pag1compared to wild-type, whichexplained the dwarfed and compact phenotype. pag1showed a de-etiolated phenotype in thedark, and exogenous application of brassinolide (BL) can rescue the growth of pag1,suggesting that pag1is a BR deficiency mutant but not a BR insensitive mutant.2. Genetic analysis showed that the dwarf phenotype was controlled by one single dominant gene,and co-segregated with the T-DNA tag. Future research proved that activation tag insertupstream of one P450gene and caused its over-expression. Ectopic expression of this gene(PAG1) in Arabidopsis caused severely dwarfed phenotypes, which can be restored by BLtreatment. Just like its homolog CYP734A1, PAG1was also up-regulated by exogenouslyapplied BL and light, which implied that PAG1may catabolize active BRs in a similar manner.3. In the constitutive expression transgenic lines, the plant height is inversely proportional toPAG1expression level, which indicated that manipulation of PAG1expression can be used tocontrol plant height. The transgenic rice lines also showed dwarf stature, which suggested thatPAG1can also be used change monocot crop height. Tissue-specific treatment and grafingexperiment showed that BR can not transport over long distance and green-tissue specificpromoter drived PAG1can reduce the plant height but only slightly effect reproductive tissues,which suggested that Spatiotemporal manipulation of PAG1expression is a promising meansof improving plant architecture. 4. Endogenous levels of BRs are mainly regulated through tissue-specific biosynthesis andcatabolism in order to maintain optimal plant growth and development. PAG1is highlyexpressed at fiber initiation and elongation stage, indicating PAG1play vital role in regulatingfiber development. The fiber of pag1is shorter than wild-type and can BL treatment can rescueits fiber elongation, which suggested that the over-expression of PAG1metabolized active BRsand effected fiber elongation. Transcriptome analysis demonstrated that PAG1plays crucialroles in regulating fiber development via controlling the level of endogenous bioactive BRs,which may affect ethylene signaling cascade by mediating VLCFA. Therefore BR may be avery important regulator of fiber elongation, a role which may be linked to effects on VLCFAbiosynthesis, ethylene signaling, the cell wall, and cytoskeleton-related genes expression.更多还原