【作者】 吴晓霞；

【导师】 梁建生；

【作者基本信息】 扬州大学 ， 作物遗传育种学， 2008， 博士

【摘要】 根系是植物体的重要组成部分,主要执行着吸收土壤养分和水分的功能。侧根的发育及形态与根系建成密切相关,生长素对侧根的发育具有重要的调节作用。有研究表明,异三聚体G蛋白在生长素介导的拟南芥侧根发育过程中发挥作用,但是其作用机理尚不明晰。本文以拟南芥异三聚体G蛋白相关亚基突变体及过表达的转基因植株与悬浮培养细胞系为材料,应用植物解剖学、植物生理学、分子遗传学等研究手段,探讨了异三聚体G蛋白在生长素介导侧根发育中的作用机理。研究结果表明:1. G蛋白相关亚基突变显著影响根系发育表型。agb1-2及gpa1agb1幼苗的侧根发生数均极显著多于野生型(Col),主根长度也显著长于Col。gpa1-3与Col间主根长度无显著性差异,但是gpa1-3幼苗的侧根发生数显著少于Col。2. G蛋白突变体的侧根发生依赖于生长素途径的调节。抑制生长素极性运输条件下,外源生长素可显著增加突变体及野生型的侧根发生数。但各基因型对生长素诱导侧根发生的敏感性存在差异,与Col相比,gpa1-3侧根发生对生长素低敏感,agb1-2及gpa1agb1对生长素超敏感。3.对种子萌发后2-4d内各基因型侧根原基发育时期统计结果表明,萌发后2d,各基因型侧根原基开始发生,gpa1-3与agb1-2的侧根原基数差异不明显,但均显著高于Col。萌发后4d,当茎端生长素开始向根部运输时,gpa1-3幼苗的侧根及侧根原基总数与Col类似,但是显著低于agb1-2和gpa1agb1。对侧根原基分布时期观察表明,gpa1-3的侧根及侧根原基约70%处于侧根发生的第3-5期,而agb1-2和gpa1agb1的侧根原基主要集中于第5-7期(agb1-2,55%;gpa1agb1,72%),Col的侧根原基在侧根发育的各个阶段均匀分布。4.种子萌发4d时去除根茎交界处以上部分处理显著抑制各基因型的侧根发生,且各基因型间侧根发生数无显著差异,说明各基因型的侧根发生均依赖于地上部茎端合成的生长素。侧根发生早期(萌发后4d)去根尖处理促进了各基因型的侧根发生,但对agb1-2侧根发生影响较小,侧根发生数的增加显著低于Col。种子萌发后7d对幼苗去根尖处理的效应则相反,gpa1-3侧根发生数的增加显著低于Col。根据影响侧根发生的根部生长素运输特征分析,根部生长素的向基运输是影响G蛋白突变体侧根发生差异的主要因素。5.对GPA1和AGB1过表达转基因幼苗G蛋白亚基定位和侧根发育特征的研究结果表明,GPA1主要定位在根尖顶端及中柱部位,而AGB1在整个根部均有分布。GPA1过表达植株的侧根发生数目较野生型显著增加,AGB1过表达植株的侧根发生数与野生型相比无显著性差异。结合G蛋白突变体根系发育特征,根据G蛋白信号转导模式,推测AGB1在侧根发育过程中具有负调节作用。对种子萌发4d内过表达株系及野生型的侧根发育时期分布规律分析表明,在萌发第4d时AGB1过表达幼苗的侧根原基主要分布于第3-4期,GPA1过表达幼苗的侧根原基总数高于Col野生型,其增加的侧根原基主要集中在第3-5期,说明GPA1过表达有利于促进侧根原基的早期形成及分裂过程。6.不同基因型悬浮培养细胞对2,4-D和NAA的响应存在显著的差异。用2μM 2,4-D/NAA处理悬浮培养细胞系,agb1-2和GPA1过表达悬浮细胞对2,4-D促进的细胞分裂不敏感,对NAA诱导的细胞伸长过程超敏感。RT-PCR结果表明,在无外源生长素处理条件下,agb1-2悬浮细胞中细胞分裂周期素基因AtCYCD4;1的表达量相对较高。NAA处理促进了agb1-2和GPA1过表达悬浮细胞中与细胞伸长相关的ABP1的表达。根据2,4-D和NAA进出细胞机制的不同,可以推测AGB1突变体及GPA1过表达细胞系具有较强的生长素极性输出能力。7.对不同基因型的根系向重性反应研究表明:突变体agb1-2和过表达GPA1植株对向重性刺激的反应显著强于Col和gpa1-3,而过表达AGB1植株的向重性反应显著低于Col。8. Real-time PCR研究结果表明,agb1-2及GPA1过表达植株根部生长素极性输出载体PIN1的表达量显著增加。根据以上研究结果,认为G蛋白突变体的侧根发生差异是由于影响了根部生长素的向基运输途径,AGB1对这一过程具有负调节作用。AGB1突变促进了根系中生长素极性输出载体PIN1转录水平的表达,从而负调节根系中生长素的向基极性输出途径,促进侧根大量发生。更多还原

【Abstract】 Roots systems, as one of the major organs of plant, perform essential tasks of absorbing water and nutrients from soil, and anchoring plants. The development and morphology of lateral roots play a crucial role in the construction of root systems. A lot of studies have shown that plant hormone auxin plays vital roles in the development of lateral roots. Recent studies showed that the heterotrimeric G-protein (G protein) is involved in auxin-dependent lateral root development. However, at present, the physiological and molecular mechanisms concerning G protein-mediated auxin-dependent lateral root development are largely unknown. In present study, we investigated the mechanisms of Arabidopsis heterotrimeric G-protein in the regulation of auxin-dependent lateral root development by using heterotrimeric G-proteinα/βsubunit null mutants and the transgenic cells and plants of overexpressed GPA1 /AGB1 plants as experimental materials with different approaches. The results showed that1. Mutation of the heterotrimeric G-proteinαsubunit andβsubunit affected significantly lateral root development of Arabidopsis. The lateral root numbers (LRNs) and the primary root length of G-proteinβsubunit mutant (agb1-2) and double mutant (gpa1agb1) were greater significantly than those of Col. No obvious difference was observed in main root length between G-proteinαsubunit mutant (gpa1-3) and wildtype, while the LRNs of gpa1-3 mutant were significantly lower than those of Col.2. The lateral root development was markedly affected by auxin. Under the condition of inhibiting auxin polar transport by using TIBA, an auxin polar transport inhibitor, exogenous IAA significantly increased the number of emerging lateral roots in heterotrimeric G protein mutants and wild type, which means that lateral roots formation in G protein mutants is also auxin-depend. However, significant differences were observed in the responses of lateral roots formation to auxin treatment among different genotypes investigated. As compared with Col, the lateral roots formation of gpa1-3 mutant was relatively insensitive and agb1-2 and gpa1agb1double mutant super-insensitive to auxin treatment3. The initiation of lateral roots occurred about at day 2 after seed germination and no obvious difference was detected in the numbers of lateral root primordia (LRP) between gpa1-3 and agb1-2 mutant, but all were significantly higher than that of Col. After 4 days of germination, when the auxin formed in the shoots began to transport to roots, the total number of lateral roots (LRs) and LRPs of gpa1-3 mutant was similar to that of Col, but significantly lower than that of gpa1agb1. Furthermore, About 70% of LRPs of gpa1-3 after 4 days germination were at 3-5 stages, while most of LRPs of agb1-2 and gpa1agb1 were at 5-7stages in agb1-2 and gpa1agb1.4. In the case of aerial tissue removal at 4 days after germination (DAG), the relative number of emerging and fully developed LRs decreased drastically compared to the intact seedlings, and no differences were observed among genotypes investigated. At the earlier stage of lateral root development (about 4 days after seed germination), excision of root tips (primary roots) significantly increased LRs except agb1-2 mutant. However, when root tips were excised after 7 days of germination, the increased LRs of mutant gpa1-3 was significantly lower than Col and other genotypes.5. Protein localization analysis using GFP fusion protein technique showed that GPA1 was mainly located in root apical and pericycle parts while AGB1 was distributed through the whole root. The density of LRs in GPA1 overexpressed seedlings was significantly higher than that of wild type plants, while no significant difference in LRNs was observed between AGB1 overexpressed seedlings and Col. The development of LRs of AGB1 overexpressed seedlings were less response to exogenous auxin and hypersensitive to TIBA while the response to auxin and TIBA in GPA1 overexpressed seedlings was similar to Col. The LRPs of AGB1 overexpressed seedlings at 4 days after germination were mainly distributed at stages 3-4, while LRPs in GPA1 overexpressed seedlings were mainly distributed at stages 3-5.6. There were significantly differences in cell division and cell elongation responses to 2,4-dichlorophenoxyacetic acid (2,4-D) andα-naphthalene acetic acid (NAA). The division of agb1-2 and GPA1-overexpressed cells was insensitive to 2,4-D (2μM) treatment and cell elongation of those genotypes was super-sensitive to NAA (2μM) treatment. RT-PCR result showed that, cell cyclin gene AtCYCD4;1 was highly expressed in agb1-2 suspension cells without exogenous auxin treatment. Gene ABP1, which was related to cell elongation, was highly expressed in agb1-2 and GPA1-overexpressed suspension cells treated with NAA. According to the different transport patyway of 2,4-D and NAA, it could be concluded that agb1-2 mutate and GPA1-overexpressed promoted auxin export capacity and enhanced the cell mitosis ability.7. Responsive to gravistimulation shown that agb1-2, GPA1-overexpressed seedlings were more responsive to gravistimulation than Col and gpa1-3 while AGB1- overexpressed seedlings were less responsive than the wild type Col.8. Real-time PCR analysis showed that the expression of PIN1 was significantly stimulated in roots of agb1-2 and GPA1 overespressed seedlings.Based on the results above, it was suggested that G protein-mediated lateral root development was largely through affecting auxin basipetal transport in roots. AGB1 negatively regulated this process by inhibiting the expression of PIN1, as a result, more lateral roots were formed in agb1-2 mutant.更多还原