DPC浸种促进棉花侧根发育的激素机制及结合态ABAscFv的制备

发布时间：2018-01-01 12:27

本文关键词：DPC浸种促进棉花侧根发育的激素机制及结合态ABAscFv的制备　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：缩节安(N,N-Dimethylpiperidinium choride,DPC)是一种在棉花上广泛使用的植物生长调节剂,其主要的生理效应之一是促进侧根发育。植物侧根发育的好坏与营养物质的吸收及抵抗低温、干旱等逆境的能力有重要的关系。本研究以DPC浸种处理棉花种子,探讨DPC促进棉花幼苗侧根发育的激素机制。主要结果如下:1.DPC浸种处理后,增加了侧根原基和侧根的数量,处理后4-7d,分别比对照增加了 32.1%、34.9%、21.8%和10.9%。同时通过石蜡切片观察到DPC浸种处理后侧根原基的起始阶段提前。2.DPC浸种处理后,IAA含量在根和子叶中表现出部位差异性。处理后2和3d,显著增加了根中的IAA含量,但降低了在子叶中的IAA和IAA结合态含量。通过免疫组化定位技术,发现DPC浸种处理后,增加了主根中IAA在中柱的积累,对表皮和皮层没有影响。3.DPC浸种处理后IAA主要合成基因GhTAA1、GhTAR2的表达量没有显著变化,但子叶中IAA结合态水解酶基因GhILR1、GhIhR3.1和GhIAR3.2的表达量上调,从而加速了 IAA结合态的水解,同时IAA输出载体GhPIN1s和GhPIN2基因表达量上调,但对IAA输入载体GhAUX1基因表达量的调节没有规律。因此推断根中自由态IAA的增加主要来自于子叶中IAA结合态的水解并通过PIN蛋白将IAA转运。4.GA3浸种处理的表型与DPC处理后的表型相反,显著降低了侧根原基数量和密度。DPC浸种处理后根中GA含量降低。GA合成基因GhCPS、GhKS、Gh20oxr1和Gh20ox2总体呈下调的趋势,GA代谢基因Gh2ox1和Gh2ox2呈上调的趋势。5.克隆了 DPC处理后表达量上调较大的IAA结合态的水解酶GhIAR3.1基因并进行功能验证,将纯化的重组蛋白GST-GhIAR3.1与IAA氨基酸结合态孵育,通过HPLC检测表明GST-GhIAR3.1能够催化IAA-Ala与IAA-Asp生成自由态IAA,其平均水解速率分别为11.6±1.9和1.86±0.25μmolIAAreleased/min/mg,该酶不能催化IAA-Ile和IAA-Phe。通过酶促动力学分析,GST-GhIAR3.1 与 IAA-Ala 的 Vmax 和Kw 计算分别为 17.2 μmol/min/mg 和 320.67 μM。6.通过载体上自带的Thromin酶将标签GST和目的蛋白GhIAR3.1分离。将目的蛋白GhIAR3.1免疫新西兰大耳白兔和Balb/c小鼠,成功制备了兔多抗和小鼠单克隆抗体并建立双抗夹心ELISA。用所建立的双抗夹心ELISA测定了子叶中水解酶GhIAR3.1的含量,发现DPC浸种处理后2d,子叶中水解酶GhIAR3.1的含量比对照增加了 17.8%。本论文还以实验室保存的ABA结合态杂交瘤细胞为材料克隆了 ABA结合态VH、VL基因,将VH、VL用一条短肽连接为scFv,scFv与MBP标签融合纯化分析其结合特性,通过ELISA表明能够结合ABA及ABA结合态。ABA、ABA-ME和ABAGE四乙酰基的10%抑制率分别为19.7、8.1和4.0 μg/ml。ABA-ME和ABAGE四乙酰基的抑制率分别为ABA之间1.7和2.4倍。将ABA结合态的scFv建立3D同源模型,并与ABA、ABA-ME和ABAGE分子对接,结果显示与ABA、ABA-ME 和 ABAGE的结合能量分别为-37.1-69.6 和-112.0 kcal.mol-1,ABA-ME/ABA和ABAGE/ABA的能量比率分别为1.87和3.0,与ELISA得出的结果一致。
[Abstract]:DPC (N, N-Dimethylpiperidinium, Choride, DPC) is a widely used in cotton plant growth regulator, one of the main physiological effects is to promote the development of lateral roots. The absorption and low temperature resistance and good nutrition plant lateral root development, has an important bearing ability of drought. In this study, DPC soaking treatment of cotton seeds, DPC on hormone mechanism of cotton seedling lateral root development. The main results are as follows: 1.DPC treatment, increased the number of lateral roots and lateral roots, 4-7d after treatment were increased by 32.1%, 34.9%, 21.8% and 10.9%. were observed by paraffin section and DPC soaking after initiation of lateral root primordia early.2.DPC treatment, IAA content in roots and cotyledons showed part difference. After treatment of 2 and 3D, significantly increased the IAA content in roots, but decreased in the cotyledon in IAA and I AA boundforms. Through immunohistochemical localization technology, found that DPC treatment, increased IAA in taproot column accumulation did not affect.3.DPC treatment IAA synthetic gene GhTAA1 on the epidermis and cortex, the expression of GhTAR2 did not change significantly, but in the cotyledon IAA binding state of GhILR1 hydrolase gene expression. The amount of up regulation of GhIhR3.1 and GhIAR3.2, thus accelerating the hydrolysis of IAA bound, while the IAA output vector GhPIN1s and GhPIN2 gene expression, but the input to the IAA expression vector of GhAUX1 gene in the regulation of the volume without law. Therefore conclude that increasing the free state in the roots of IAA mainly from cotyledon IAA bound hydrolysis by PIN the phenotype of DPC protein and IAA translocation of.4.GA3 after soaking in contrast, decreased the content of GA in the root of lateral root primordium number and density of.DPC treatment decreased.GA synthesis gene GhCPS, GhKS, Gh20oxr1 and Gh20ox2 showed an overall downward trend, the metabolism of GA gene Gh2ox1 and Gh2ox2 was higher in.5. was cloned after DPC treatment up-regulated IAA hydrolase GhIAR3.1 gene combined with large state and functional verification, the purified recombinant protein GST-GhIAR3.1 and IAA amino acid bound incubation by HPLC detection showed that GST-GhIAR3.1 IAA-Ala and IAA-Asp can catalyze the formation of free state IAA, the average rate of hydrolysis were 11.6 + 1.9 and 1.86 + 0.25 molIAAreleased/min/mg, IAA-Ile and IAA-Phe. of the enzyme catalyzed not by enzymatic kinetic analysis, Vmax and Kw to calculate GST-GhIAR3.1 and IAA-Ala were Thromin 17.2 mol/min/mg and 320.67 M.6. by the carrier on its own the label GST and GhIAR3.1 protein. GhIAR3.1 protein separation to immunize New Zealand white rabbits and Balb/c mice, rabbit were prepared. And anti mouse monoclonal antibody and establishment of double antibody sandwich ELISA. content in cotyledon hydrolase GhIAR3.1 was determined by the double antibody sandwich ELISA, found that DPC treatment 2D content in cotyledon hydrolase GhIAR3.1 increased by 17.8%. of the laboratory preserved ABA as material to clone the ABA bound VH states hybridoma cells with VL gene, VH, VL with a short peptide connection for scFv, purification and analysis of its binding properties of scFv and MBP fusion tag, by ELISA that can be combined with ABA and ABA combined with.ABA ABA-ME and ABAGE state, four acetyl 10% inhibition rates were 19.7,8.1 and 4 g/ml.ABA-ME and ABAGE four deacetylation rate was ABA between 1.7 and 2.4 times. The ABA bound scFv 3D homology models, and with ABA, ABA-ME and ABAGE molecular docking, with that of the ABA, the binding energy ABA-ME and ABAGE respectively. -37.1-69. The energy ratios of 6 and -112.0 kcal.mol-1, ABA-ME/ABA and ABAGE/ABA are 1.87 and 3, respectively, which are in accordance with the results obtained from ELISA.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S562

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