Sasm05菌株提高东南景天锌富集的作用机制研究

发布时间：2018-03-09 22:11

本文选题：超积累植物　切入点：锌　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：超积累生态型东南景天(Sedum alfredii Hance)是我国原生的锌(Zn)/镉(Cd)超积累植物,对重金属污染土壤具有较好的修复潜力,但其生物量较小,修复效率亟需进一步提高。近年来,利用植物促生菌提高植物修复效率已成为国内外研究的热点。本研究以东南景天及其茎中分离的特异内生菌荧光假单胞杆菌Sasm05为材料,通过溶液培养试验,采用三亲结合法、激光共聚焦显微镜分析、电感耦合等离子体质谱分析(ICP-MS)、实时定量PCR(q-RT-PCR)等技术研究了强化接种Sasm05对东南景天锌吸收与转运的影响及其生理机制,取得的主要研究成果如下:1、通过三亲结合法对Sasm05进行了绿色荧光蛋白(GFP)标记,利用激光共聚焦显微镜观察发现强化接种的Sasm05进入到了植物的根表、根内和茎中。随着Zn处理浓度的增加,东南景天地上部与根系的生物量与Zn浓度均显著增加。接种Sasm05后,地上部生物量比未接种对照增加了 37.3%-59.2%,根系生物量增加了 58%-108%;地上部的Zn浓度增加了 16.7%-54.7%,根系的Zn浓度增加了 37%-40.6%;叶绿素含量增加了 35.5%-56.2%。同时,Sasm05显著增加了 HMA、Nramp和ZIP等转运蛋白基因的表达量。2、研究了强化接种Sasm05对东南景天锌吸收转运动力学的影响,发现随着培养时间的增加,植物生物量显著增加;在200 μMZn处理浓度下,30 d时地上部和根系的生物量增幅最大,分别为262%和421%;接种Sasm05进一步提高了地上部和根系的生物量,增幅为34.5%和46.8%。相比未接种对照,Sasm05提高了植物体内的Zn浓度(9.2%-120%),叶绿素含量(15.3%-69.8%)以及金属离子转运蛋白基因的表达量,其中根系Nramp1的表达量增加了 2.5-34.2 倍。3、比较了强化接种Sasm05和IAA处理对东南景天根系发育和锌吸收转运的作用效应。发现IAA对根系生物量的影响主要表现为低浓度促进,高浓度抑制,确定促生效应最佳的IAA浓度为11μM。Sasm05和IAA均可提高地上部锌的积累量,其中Sasm05显著增加了植物地上部的生物量,其促生效应比IAA更强;并且可促进Zn、Ca、Fe、Mg、Mn、Cu等金属离子在植物植物地上部和根系的浓度,而IAA作用效果不明显。进一步分析发现,Sasm05与IAA均影响Nramp1和IRT1等转运基因的表达,但两者的作用效应并不一致,说明Sasm05不仅可能通过分泌IAA发挥促生作用,还可能直接影响相关基因的表达进而调节重金属的吸收运输,从而调控植物体内的重金属浓度以达到提高东南景天锌富集的效应。
[Abstract]:The superaccumulative ecotype Sedum alfredii Hanceis a native zinc-zn-zn / CD / CD superaccumulant plant in China, which has good remediation potential for heavy metal contaminated soils, but its biomass is small, and its remediation efficiency needs to be further improved in recent years. It has become a hot topic to improve the efficiency of phytoremediation by plant growth promoting bacteria. In this study, Sasm05, a specific endophytic bacterium isolated from Sedum and its stems, was used as the material, and the method of three-amphiphilic combination was used in the solution culture experiment. Laser confocal microscopy (LCM), inductively coupled plasma mass spectrometry (ICP-MS) and real-time quantitative PCRQ- RT-PCR were used to study the effects of enhanced inoculation of Sasm05 on zinc absorption and transport and its physiological mechanism. The main research results were as follows: 1. Sasm05 was labeled with green fluorescent protein (GFP) by the three-amphiphilic binding method. Using confocal laser microscopy, it was found that the Sasm05 inoculated with enhanced inoculation entered the root surface of the plant. With the increase of Zn concentration, the biomass and Zn concentration of shoot and root increased significantly. After inoculation with Sasm05, the biomass and Zn concentration of shoot and root increased significantly. The aboveground biomass increased by 37.3-59.2 and the root biomass increased by 58-108. the Zn concentration in the shoot increased 16.7-54.7, the root Zn concentration increased 37-40.6. the chlorophyll content increased 35.5-56.2.The Sasm05 significantly increased the HMANramp. And the expression of ZIP and other transporter genes. 2. The effect of Sasm05 inoculation on zinc uptake and transport kinetics was studied. The results showed that the biomass of plant increased significantly with the increase of culture time, and the biomass of shoot and root increased most at the concentration of 200 渭 MZn for 30 days, which were 262% and 421, respectively, and inoculated with Sasm05 further increased the biomass of shoot and root. The increases were 34.5% and 46.8. Compared with the uninoculated control, Sasm05 increased the Zn concentration in the plant and the content of chlorophyll in the plant, the chlorophyll content was 15.3- 69.8%) and the expression of the metal ion transporter gene. The expression of Nramp1 in roots increased by 2.5-34.2 times. The effects of Sasm05 and IAA on root development and zinc uptake and transport were compared. It was found that the effect of IAA on root biomass was mainly promoted by low concentration. The best concentration of IAA, 11 渭 M. Sasm05 and IAA, could increase the accumulation of Zn in shoot, and Sasm05 significantly increased the biomass of shoot, which should be stronger than IAA. In addition, the concentration of metal ions such as IAA in plant shoots and roots was promoted, but the effect of IAA was not obvious. It was found that both Sasm05 and IAA affected the expression of Nramp1 and IRT1 genes, but the effects were not consistent. It is suggested that Sasm05 may not only promote growth by secreting IAA, but also directly affect the expression of related genes and regulate the absorption and transport of heavy metals, thus regulating the concentration of heavy metals in plants so as to increase the concentration of zinc in Sedum southeastern China.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X17;X53

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