【作者】 丁硕；

【导师】 安黎哲；

【作者基本信息】 兰州大学 ， 植物学， 2011， 博士

【摘要】 植物内生细菌是定殖于健康的植物组织内部,不引起宿主植物表现出外在病症的一类细菌。一些植物内生细菌可以提高植物对非生物胁迫和生物胁迫如盐胁迫、干旱胁迫、低温胁迫、金属离子胁迫和病虫害等的抗性,促进宿主植物的生长。研究发现,高山冰缘植物中含有大量内生细菌,且部分内生细菌有抗冻、固氮、溶解难溶性磷、产IAA等特性,具有潜在的生物学和生态学作用。高山离子芥(Chorispora bungeana Fisch.& C.A. Mey)是一种典型的高山冰缘植物,具有很强的抗寒能力,是研究植物抗寒机制的理想材料。为了探究高山冰缘植物内生细菌对其宿主植物抗寒性的影响,本论文以高山离子芥为材料,研究并阐释了内生细菌对高山离子芥抗寒性的影响及其机制,试图为低温胁迫下植物内生细菌的应用和植物抗寒机制的研究提供实验依据和理论基础。获得的主要研究结果如下：1.确定了7株高山冰缘植物内生细菌在高山离子芥再生植株体内的定殖能力,选择冰缘植物内生优势菌株Enfl2和Enf30进行抗寒机制的研究。对7株高山冰缘植物内生细菌在高山离子芥再生植株体内的定殖能力进行检测,结果表明,不论在正常环境中(20℃)还是低温胁迫(0℃)下,除Enf42 (Sphinqomonas sp.)外,其余6株内生细菌Enf10 (Sanguibacter sp.)、Enf69 (Plantibacter sp.)、Enf11 (Rhodococcus sp.)、Enf12 (Clavibacter sp.)、Enf30 (Pseadomonas sp.)和Ens37 (Pseadomonas sp.)均可成功定殖于高山离子芥再生植株并在其体内传导。综合分析后选择冰缘植物内生优势菌株Enf12和Enf30进行抗寒机制的研究。2.阐明了低温胁迫下Enf12对维持植物细胞膜结构的完整性和稳定性发挥作用,进而提高高山离子芥的抗寒能力。测定结果表明,Enf12明显降低了低温胁迫下高山离子芥再生植株和悬浮细胞的离子渗漏率及MDA含量,说明Enf12在低温下维持植物细胞膜结构的完整性和稳定性方面发挥积极作用,从而提高高山离子芥的抗寒能力。3.揭示了内生细菌Enf12通过提高植物的抗氧化防御能力,抑制ROS过量积累,降低细胞氧化损伤,从而提高高山离子芥抗寒性。研究表明,与未接菌植株和悬浮细胞相比,接种Enf12会明显提高低温胁迫下高山离子芥再生植株和悬浮细胞的抗氧化物酶SOD、CAT、POD和APX活性,诱导脯氨酸含量明显上升,显著抑制低温诱导的ROS(H2O2和O2-)过量积累,以降低ROS过量积累对细胞造成的氧化胁迫和氧化损伤,从而提高高山离子芥对低温胁迫的抵抗能力。进一步揭示了内生细菌对高山离子芥抗寒性的影响及其机制。4.对高山离子芥悬浮细胞超微结构的观察分析表明,与未接菌细胞相比,接种Enf12可以减轻低温胁迫(4℃)对悬浮细胞质体造成的损伤,提高细胞对低温的耐受力,增强高山离子芥悬浮细胞的抗寒能力。5.阐明了Enf12能够促进高山离子芥再生植株生长。生物量测定结果表明,与未接菌植株相比,接种高山离子芥内生细菌Enf12的植株在常温(20℃)和低温胁迫(0℃)下,其地上部分生物量均显著增高,说明Enf12可以促进高山离子芥再生植株生长。6.本论文同时研究了接种Enf30的高山离子芥再生植株和悬浮细胞,发现Enf30对高山离子芥抗寒性及其生长无明显影响。综上所述,内生细菌Enf12和Enf30能够定殖于高山离子芥体内,Enf12可以增加高山离子芥的生长量,低温胁迫下对其细胞结构起保护作用,提高高山离子芥抗寒性,而Enf30对高山离子芥抗寒性及其生长无明显影响。本研究阐明了高山冰缘植物内生细菌Enf12与高山离子芥抗寒性的关系,进一步揭示了高山离子芥适应低温环境的生理生态机制。更多还原

【Abstract】 Endophytic bacteria can be defined as those bacteria that colonize the internal tissue of the plant showing no external sign of infection or negative effect on their host. Some of them have been shown to enhance plant growth and increase resistance against biotic stress and tolerance to abiotic stress in many plants, including salt injury, chilling stress, water deficit, metal stress, pathogen infection and plant diseases,. The objective of this study was to evaluate the effect of the endophytic bacteria, which were isolated from alpine-subnival plants, on chilling tolerance of host plants and demonstrate the mechanism. We used Chorispora bungeana as research material in this study, which is a representative alpine-subnival plant. Physiological markers for chilling stress were assessed in regenerated plantlets and suspension cultured cells of Chorispora bungeana subjected to chilling stress (0℃or 4℃). The major results are as follows:1. In the present study 7 strains of endophytic bacteria except Sphinqomonas sp. strain Enf42 were capable of colonizing internal tissues of regenerated plantlets of C. bungeana and moved in them under both normal (20℃) and chilling (0℃) conditions. The 7 strains of endophytic bacteria were Sanguibacter sp. strain Enf10, Plantibacter sp. strain Enf69, Rhodococcus sp. strain Enf11, Clavibacter sp. strain Enf12, Pseadomonas sp. strain Enf30, Pseadomonas sp. strain Ens37 and Sphinqomonas sp. strain Enf42. Based on comprehensive analysis we selected endophytic bacteria strains Enf12 and Enf30 for further study.2. Enf12 significantly decreased electrolyte leakage and content of MDA in regenerated plantlets and suspension cultured cells of Chorispora bungeana exposed to chilling stress, indicating that the strain Enf12 may have the potential to enhance stability of biological membrane systems and lower the sensitivity of Chorispora bungeana to chilling injury.3. During chilling treatment, Enf12 inoculation led to further increases in activities of antioxidant enzymes (SOD, CAT, POD and APX) and proline accumulation, and significantly lower levels of ROS (H2O2 and O2-) in the plantlets and suspension cultures. Endophytic bacteria strains Enf12 appears to confer chilling tolerance on Chorispora bungeana by increasing the activities of antioxidant enzymes, which could limit ROS levels as well as lipid peroxidation, thus alleviating chilling-induced oxidative stress and damage.4. Ultrastructure of suspension cultures showed that Enf12 inoculation alleviated the damage in plastid resulted from chilling stress. It may indicate that the endophyte Enf12 improved the tolerance of Chorispora bungeana to low temperature.5. Compared with the non-inoculated plantlets, the aerial part biomass of Enf12-inoculated plantlets was significantly greater under both normal (20℃) and chilling (0℃) conditions, indicating that Enf12 has the ability to enhance the growth of Chorispora bungeana plantlets.6. The study of Enf30-inoculated regenerated plantlets and suspension cultured cells of Chorispora bungeana showed that Enf30 have no significant effect on the growth and tolerance of Chorispora bungeana to chilling stress.Thus, our results demonstrate that the strain Enf12 and Enf30 are capable of colonizing internal tissues of C. bungeana. Enf12 inoculation stimulates the growth of C. bungeana and improves its tolerance to chilling stress through enhancing the antioxidant defense system. However, Enf30 have no significant effect on the growth and tolerance of Chorispora bungeana to chilling stress.更多还原