【作者】 杨艳；

【导师】 唐明；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2010， 硕士

【摘要】 本文采用纯培养技术筛选出一株抗旱性强的外生菌根真菌,测定其在不同干旱胁迫下菌丝体分泌植物激素的差异;采用盆栽法接种油松苗,研究聚乙二醇-6000(PEG-6000)模拟干旱胁迫下,菌根苗和非菌根苗生长和生理生化指标的变化,分析干旱胁迫下,菌根真菌、菌根苗、非菌根苗蛋白表达差异,揭示外生菌根真菌提高油松苗的抗旱机制。得出以下主要结论:1.抗旱菌株的筛选对纯培养条件下3种外生菌根真菌生长特性研究表明,干旱胁迫只改变了外生菌根真菌生长速度和生物量积累,对增长模式没有影响。轻度干旱胁迫(10% PEG浓度)对3种外生菌根真菌有一定的促生长作用,随着干旱胁迫的加剧(15%~35% PEG浓度),3种外生菌根真菌的抑菌率都增大,抗旱性由强到弱依次为灰环粘盖牛肝菌(Suillus laricinus(Berk.in Hook.)O.Kumtze)>绒粘盖牛肝菌(Suillus tomentosus(Kauff.)Sing.Snell & Dick)>灰鹅膏菌(Aminita vaginata(Bull.:Fr.)Vitt.)。2. PEG胁迫下抗旱菌株产生植物激素的差异不同PEG浓度干旱胁迫下,液体培养灰环粘盖牛肝菌,发现当PEG胁迫浓度由0%增加到25%时,菌丝体中赤霉素含量由26.53μg/g递减到0μg/g,生长素含量由180.98μg/g递减到0μg/g。15% PEG浓度下脱落酸积累最多,为184.36μg/g,当干旱胁迫超过其忍耐程度(20% PEG浓度),抑制脱落酸的积累。3.外生菌根真菌对油松的接种效应正常水分下,接种菌根真菌(灰环粘盖牛肝菌)促进油松苗高、茎粗和根长,提高地上干重、地下干重和根冠比(P<0.05),菌根苗比非菌根苗分别提高了26.36%、8.47%、24.83%、90.91%、175%和36.84%,二者相对含水量差异不显著(P>0.05)。菌根依赖性(MD)大于200%,属于中等依赖性。菌根苗和非菌根苗生理指标无显著性差异(P>0.05)。4. PEG胁迫下外生菌根真菌对油松抗氧化酶活性的影响PEG模拟干旱胁迫下测定菌根苗和非菌根苗根系SOD和CAT活性,研究表明:随着胁迫时间的延长,菌根苗和非菌根苗SOD活性、30% PEG浓度下的CAT活性呈“降低-升高-降低”的变化趋势;0%、10%和20%浓度下CAT活性呈“升高-降低”的变化趋势。相同PEG浓度胁迫1、2和4 h,菌根苗SOD活性显著高于非菌根苗活性(P<0.05);胁迫8和12 h差异不显著(P>0.05)。胁迫2 h,20% PEG浓度下CAT活性增幅(与胁迫1 h相比)最大,菌根苗增加了3.20倍,非菌根苗增加了2.84倍,菌根苗酶活性比非菌根苗高69.50%。5. PEG胁迫下外生菌根真菌对油松丙二醛(MDA)含量及质膜透性的影响PEG模拟干旱胁迫下测定菌根苗和非菌根苗根系MDA含量和根系电导率,研究表明:随着胁迫时间的延长,菌根苗和非菌根苗MDA含量逐渐增加,根系电导率呈波动性变化。相同PEG浓度胁迫1 h,菌根苗和非菌根苗MDA含量无显著性差异(P>0.05),胁迫2~12 h,菌根苗MDA含量显著低于非菌根苗(P<0.05)。30% PEG浓度胁迫12 h MDA达最大值,非菌根苗比菌根苗高38.13%。相同PEG浓度胁迫相同的时间,非菌根苗电导率均大于菌根苗的电导率。6. PEG胁迫下外生菌根真菌对油松脯氨酸(Pro)含量的影响PEG模拟干旱胁迫下测定菌根苗和非菌根苗根系Pro含量,研究表明:随着胁迫时间的延长,菌根苗和非菌根苗Pro含量呈“升高-降低”的变化趋势。相同PEG浓度胁迫1~2 h,菌根苗Pro含量高于非菌根苗;胁迫4~12 h,菌根苗Pro含量低于非菌根苗。30% PEG浓度胁迫4 h,非菌根苗脯氨酸含量高达85.60μg/g,比菌根苗高50.12%。7. PEG胁迫下外生菌根真菌对油松萎蔫率和抗旱级别的影响PEG模拟干旱胁迫下测定菌根苗和非菌根苗根系萎蔫率和形态抗旱级别,研究表明:随着PEG浓度的增加和胁迫时间的延长,菌根苗和非菌根苗叶片萎蔫,植株倒伏,萎蔫率和抗旱级别增大。菌根苗萎蔫速度慢于非菌根苗,程度也较轻。综合分析表明,接种菌根真菌的油松苗抗旱性比未接种强。8. PEG胁迫下菌根真菌、油松苗根系蛋白表达差异由蛋白电泳图谱可知,干旱胁迫下,菌根真菌、菌根苗和非菌根苗蛋白条带不尽相同,蛋白的表达量与胁迫的强度有关。真菌菌丝体表达了分子量为66.2 KDa和28.4 KDa的蛋白,随着PEG浓度升高,蛋白表达量增多,但30% PEG浓度下没有蛋白表达。菌根苗比非菌根苗多表达了分子量为66.2 KDa的蛋白,少表达13.0 KDa的蛋白,同时提高了97.5 KDa蛋白的表达量。14.5 KDa~20.1 KDa蛋白在菌根苗和非菌根苗中无明显变化。更多还原

【Abstract】 In this paper, a drought-resistant ectomycorrhizal fungi was selected in pure culture, and its phytohormone excretion in mycelium was measured under different drought stress. Pot culture experiments were carried out to study the inoculation effects with the ECMF on the growth and some physio-biochemical indexes of Pinus tabulaeformis Carr.seedlings under drought stress using treatments of different PEG 6000 concentrations in 1/2 Hoagland liquid. In addition, protein expression in ECMF, mycorrhizal plants and non-mycorrhizal plants under different drought stress was analysed. Mechanism of the promotion of drought resistance of Pinus tabulaeformis Carr. with ECMF was revealed. The results were as follows:1. Selection for a drought-resistant ECMFThe growth characteristics of three kinds of ECMF under PEG stress in pure culture were studied, and the results showed that the growth models of the three ECMF were not affected by PEG stress, but the growth rates and biomass changed. Lower PEG concentration(10%) could stimulate the growth of them. Inhibitory efficiency became higher with the increasing PEG concentration(15%~35%). Their resistance to drought were: Suillus laricinus(Berk.in Hook.)O.Kumtze>Suillus tomentosus(Kauff.)Sing.Snell & Dick>Aminita vaginata(Bull.:Fr.)Vitt.2. Phytohormone excretion under PEG stressSuillus laricinus was cultured in MMN liquid stressed with different concentrations of PEG, and the results showed that with the increasing drought stress(0%~25%), the content of gibberellin(GA) and auxin(IAA) in mycelium reduced from 26.53μg/g to 0μg/g and 180.98μg/g to 0μg/g. S. laricinus accumulated the most abscisic acid(ABA)at 15% PEG concentration, 184.36μg/g; when drought stress was beyond the tolerance of S. laricinus(20% PEG concentration), it inhibited the accumulation of abscisic acid(ABA).3. Inoculation effects on Pinus tabulaeformis Carr.S. laricinus significantly promoted the plant hight, stem diamter, plant dry weight and root top rate of P. tabulaeformis, which in mycorrhizal plants was higher by 26.36%, 8.47%, 103.85% and 36.84% than that in non-mycorrhizal plants. And there was no significant difference between the content of relative water. The mycorrhizal dependence was more than 200%, which belonged to medium dependence.There was no significant difference between physiological index of mycorrhizal and non-mycorrhizal plants in normal.4. Effects of ECMF on antioxidant enzyme of P. tabulaeformis under PEG stress SOD and CAT activitiesof mycorrhizal and non-mycorrhizal plants under PEG stress were determined, and the results showed that with increasing duration, SOD activities of mycorrhizal and non-mycorrhizal plants, together with CAT activities at 30% PEG concentration presented descend-ascend-descend trend; CAT activities of other treatments presented ascend-descend trend. SOD activities in mycorrhizal plants were significantly higher than that in non-mycorrhizal plants at 1, 2 and 4 hour stress; and there was no significant difference between them at 8 and 12 hour stress.The highest CAT activities occurred at 2 h of 20% PEG concentration, which in mycorrhizal plants(increased 3.20 times) were higher by 69.50% than that in non-mycorrhizal plants(increased 2.84 times).5.Effects of ECMF on MDA content and membrane permeability of P. tabulaeformis under PEG stressMDA content and membrane permeability of mycorrhizal and non-mycorrhizal plants under PEG stress were determined, and the results showed that MDA content increased by degree with increasing PEG concentration and duration. Root conductivity fluctuated. MDA content between mycorrhizal and non-mycorrhizal plants had no significant difference at 1 hour stress, but it was significantly lower in mycorrhizal plants than that in non-mycorrhizal plants from 2~12 h. MDA content reached the summit at 12 h of 30% PEG concentraiton, and the content in non-mycorrhizal plants were higher by 38.13% than that in mycorrhizal plants. Relative conductivity of non-mycorrhizal plants was higher than that in mycorrhizal plants.6. Effects of ECMF on Pro content of P. tabulaeformis under PEG stressPro content of mycorrhizal and non-mycorrhizal plants under PEG stress was determined, and the results showed that Pro content presented ascend-descend trend with increasing duration. Pro content in mycorrhizal plants was higher than that in non-mycorrhizal plants from 1~2 h, and it was opposite from 4~12 h. Pro content achieved the peak at 4 h, which in non-mycorrhizal plants at 30% PEG concentration (85.60μg/g) was higher by 50.12% than that in mycorrhizal plants.7. Effects of ECMF on wilted coefficient and drought-resistant level of P. tabulaeformis under PEG stressThe wilted coefficient and drought-resistant level of mycorrhizal and non-mycorrhizal plants under PEG stress were determined, and the results showed that wilted coefficient and drought-resistant level increased with increasing PEG concentration and duration. Mycorrhizal plants reached the same damaging degree later than non-mycorrhizal plants. Or mycorrhizal plants appeared the same damaging degree under greater drought stress compared with non-mycorrhizal plants. Non-mycorrhizal plants had a faster and more serious wilting than mycorrhizal plants. In conclusion, in enhanced drought resistance of P. tabulaeformis after inoculating with ECMF.8. Protein expression of ECMF and P. tabulaeformis under PEG stressFrom the SDS-PAGE electrophoretogram, it was observed that ECMF, mycorrhizal plants and non-mycorrhizal plants had different protein bands under drought stress. Protein expression was related to drought strength. More protein(66.2 KDa and 28.4 KDa)was expressed with increasing PEG concentration in mycelium, and no protein was expressed at 30% PEG concentration. Compared with non-mycorrhizal plants, mycorrhizal plants increased the expression of 97.5 KDa protein, besides expressed a more 66.2 KDa protein, and not expressed 13.0 KDa protein. There was no significant difference for protein(14.5 KDa~20.1 KDa)between mycorrhizal and non-mycorrhizal plants.更多还原