利用原生质体融合及基因工程的方法对柑橘采后生防菌34-9遗传改良的初步研究

Preliminary Studies on Genetic Improvement of Biocontrol Yeast 34-9 of Citrus by Protoplast Fusion and Genetic Engineering

作者: 专业:园艺学 导师:邓伯勋 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

Kloeckera apiculata, genetic improvement, protoplast fusion, genetic engineering

        生防酵母菌34-9(Kloeckera apiculata)是本实验室从自然界中自主分离得到的,对柑橘青、绿霉病具有很强的拮抗作用,但因絮凝性差、生物量低、抗菌谱窄和抑菌活性有待提高等问题,限制了其商业化应用。为了提高现有生防酵母的综合性状,迫切需要对其在基因水平上进行遗传改良,同时为进一步开发出新型、安全无毒、多功能的生物保鲜剂提供理论依据。本研究对菌株34-9的遗传改良进行了初步探索。采用双亲灭活原生质体融合技术,分析了影响亲本菌株34-9和酿酒酵母原生质体制备与再生的主要因素,明确了原生质体制备和再生的最佳条件,确立了原生质体灭活条件,对融合子进行了初步筛选和鉴定。同时利用根癌农杆菌介导的转化、醋酸锂法和电转化的转化方法,对菌株34-9转化条件进行了摸索,并对转化子进行了初步鉴定。主要研究结果如下:1.利用rDNA-ITS分子鉴定,进一步证实了菌株34-9的属、种名。确定该菌株为柠檬形克勒克氏酵母(K. apiculata),是有孢汉逊酵母属(Hanseniaspora uvarum)的无性世代。2.分别确定了菌株34-9和酿酒酵母原生质体制备和再生的适宜条件。菌株34-9原生质体制备和再生的适宜条件:采用0.05 mol/L EDTA-Na2和0.5%p-巯基乙醇混合液,28℃预处理10min;培养时间为16h;酶解液浓为1.5%的蜗牛酶,酶解时间为3h;渗透压稳定剂为0.8 mol/L的KCl溶液。酿酒酵母原生质体制备和再生的适宜条件:最适产孢培养基为Kleyn;培养时间为26 h;酶解液浓度为1.5%的蜗牛酶,酶解时间为2h;渗透压稳定剂为0.8 mol/L的蔗糖溶液。3.确立了菌株34-9和酿酒酵母原生质体灭活的适宜条件。菌株34-9化学灭活的参数:0.9%的碘乙酸作用10 min;酿酒酵母热灭活参数:55℃作用15min。在28℃条件下,利用35%PEG-4000作为促融剂进行融合。4.以菌株34-9和酿酒酵母为亲本,进行原生质体双亲灭活和融合,经过菌落大小、絮凝性检测和对峙实验初步筛选出融合子RH-1,其絮凝形成速度比对照明显快且絮凝物大;并且融合子RH-1仍具有较好的抑菌效果。5.初步确定了菌株34-9电击转化的条件:对数生长期细胞,1 mol/L山梨醇处理,20μg/mL质粒DNA,电压1.5kv,电击时间5 msec,电阻200Ω,电容25μF,电击杯0.2 cm;经初步鉴定获得5株转化子,且具有很高的遗传稳定性。
    Kloeckera apiculata strain 34-9, as a postharvest biocontrol yeast, has been shown to have high antagonistic activity against blue mold and green mold caused by Penicillium italicum and P. digitatum in citrus fruit. However, the problems of poor flocculence, low biomass, narrow antagonistic spectrums and antagonistic activity remained to be improved, have limited its commercial application. Therefore, it is necessary to carry out genetic improvement on the strain 34-9, in order to enhance its comprehensive characters and obtain excellent strains. Meanwhile, it will provide a theoretical basis to explore a safe and non-toxic biological agent.The aim of this paper is to modify the genetic characters of strain 34-9. It mainly included that the fusion was completed by using the inactivated parental protoplasts, the key factors of affecting the protoplast formation and regeneration were studied, the optimum conditions for inactivation were determined and the fusants were selected and identified. It also conducted a preliminary exploration of transformation system of strain 34-9 by using Agrobacterium-mediated transformation, lithium acetate transformation and electroporation transformation. The main results were as follows:1. We further confirmed the genus and species name of strain 34-9 by using rDNA-ITS molecular identification. The strain 34-9 was identified as K. apiculata, which was the asexual generation of Hanseniaspora uvarum.2. The proper protoplast preparation and regeneration conditions of strain 34-9 and Saccharomyces cerevisiae were determined separately. The proper conditions for strain 34-9:pretreatment 10 min with 0.05 mol/L EDTA-Na2 and 0.5%β-mercapto-ethanol at 28℃, cultivation time 16 h, enzymatic concentration 1.5% snailase, enzymatic time 3 h, osmotic stabilizer 0.8 mol/L KCl; The proper conditions for S. cerevisiae:optimum sporulation medium Kleyn, cultivation time 26 h, enzymatic concentration 1.5% snailase, enzymatic time 2 h, osmotic stabilizer 0.8 mol/L sucrose.3. The proper inactivation parameter of strain 34-9 and S. cerevisiae were determined respectively. After strain 34-9 was inactivated with 0.9% iodacetic acid in 10 min and S. cerevisiae was inactivated by heat in 15 min at 55℃, they could be fused by 35% PEG-4000 at 28℃.4. Using strain 34-9 and S. cerevisiae as parent strains, after protoplast inactivation, fusion, we screened one performance markable raised fusant RH-1, by the colony size, flocculation detection and antagonistic experiment. The fusant’s flocculation formed faster than strain 34-9, which still had a good antagonistic activity.5. We preliminarily determined the electroporation conditions. The optimal electroprated stage for strain 34-9 was at intermediate logarithmic phage. Under the conditions of voltage of 1.5 kv,1 mol/L sorbitol,20μg/mL plasmid DNA, shock time 5 msec, resistance 200Ω, capacitance 25μF and 0.2 cm cuvettes, we obtained five transformants with high genetic stability.
        

利用原生质体融合及基因工程的方法对柑橘采后生防菌34-9遗传改良的初步研究

摘要7-9
Abstract9-10
1 前言11-21
    1.1 柑橘采后病害的防治策略研究11-15
        1.1.1 柑橘采后损失情况11-12
        1.1.2 柑橘采后真菌病害的防治措施12-13
        1.1.3 柑橘采后病害的拮抗菌13-15
    1.2 采后生防酵母菌的研究进展15-16
        1.2.1 采后生防酵母菌是国内外研究热点15
        1.2.2 生防酵母菌存在的问题15-16
        1.2.2 问题的解决途径16
    1.3 微生物菌种改良的方法16-17
        1.3.1 诱变育种16-17
        1.3.2 杂交育种17
        1.3.3 基因工程育种17
    1.4 原生质体融合技术在遗传改良中的应用17-19
        1.4.1 原生质体制备和再生过程中的影响因素18
        1.4.2 原生质体融合技术的应用18-19
    1.5 基因工程技术在遗传改良中的应用19-20
    1.6 课题来源及意义20-21
2 材料与方法21-35
    2.1 试验材料21-23
        2.1.1 菌株和质粒21
        2.1.2 供试培养基21
        2.1.3 主要试剂和引物21-22
        2.1.4 试验菌株保存方法22-23
        2.1.5 仪器设备23
    2.2 试验方法23-35
        2.2.1 生防酵母菌 34-9 的 rDNA-ITS 分子鉴定23-27
            2.2.1.1 生防酵母菌 34-9 DNA 提取23-24
            2.2.1.2 利用通用引物扩增rDNA基因簇ITS全序列24-25
            2.2.1.3 目的片段回收25
            2.2.1.4 PCR产物与载体连接25-26
            2.2.1.5 大肠杆菌感受态的制备26
            2.2.1.6 连接产物转化大肠杆菌26-27
            2.2.1.7 重组克隆的筛选和测序分析27
        2.2.2 生长曲线测定27
        2.2.3 原生质体融合的方法27-30
            2.2.3.1 酿酒酵母单倍体化27-28
            2.2.3.2 原生质体制备28-29
            2.2.3.3 原生质体再生29
            2.2.3.4 致死条件的确立29
            2.2.3.5 原生质体融合29-30
            2.2.3.6 融合子筛选及鉴定30
        2.2.4 基因工程的方法30-35
            2.2.4.1 菌株 34-9 对潮霉素B的敏感性检测30
            2.2.4.2 质粒的提取及鉴定30-32
            2.2.4.3 根癌农杆菌介导的转化方法32-33
            2.2.4.4 醋酸锂转化方法33
            2.2.4.5 电转化方法33-34
            2.2.4.6 转化子稳定性检测34-35
3 结果与分析35-53
    3.1 生防酵母菌 34-9 的 rDNA-ITS 序列分析35
    3.2 利用原生质体融合的方法对菌株 34-9 进行遗传改良35-48
        3.2.1 影响菌株 34-9 原生质体制备率和再生率的因素35-41
            3.2.1.1 菌株 34-9 的生长曲线35-36
            3.2.1.2 预处理对菌株 34-9 原生质体制备率和再生率的影响36
            3.2.1.3 培养时间(菌龄)对菌株 34-9 原生质体制备率和再生率的影响36-38
            3.2.1.4 酶解液浓度对菌株 34-9 原生质体制备率和再生率的影响38-39
            3.2.1.5 酶解时间对菌株 34-9 原生质体制备率和再生率的影响39-40
            3.2.1.6 渗透压稳定剂对菌株 34-9 原生质体制备率和再生率的影响40-41
        3.2.2 影响酿酒酵母原生质体制备率和再生率的因素41-47
            3.2.2.1 不同产孢培养基对酿酒酵母孢子形成的影响41-42
            3.2.2.2 酿酒酵母单倍体的生长曲线42
            3.2.2.3 培养时间(菌龄)对酿酒酵母原生质体制备率和再生率的影响42-44
            3.2.2.4 酶解液浓度对酿酒酵母原生质体制备率和再生率的影响44-45
            3.2.2.5 酶解时间对酿酒酵母原生质体制备率和再生率的影响45-46
            3.2.2.6 渗透压稳定剂对酿酒酵母原生质体制备率和再生率的影响46-47
        3.2.3 亲本致死条件47
            3.2.3.1 菌株 34-9 的致死条件47
            3.2.3.2 酿酒酵母的致死条件47
        3.2.4 融合子的获得与筛选47-48
    3.3 利用基因工程的方法对菌株 34-9 进行遗传改良48-53
        3.3.1 菌株34-9对潮霉素B的敏感性检测48-49
        3.3.2 质粒 pTFCM-hph 和 pFA6a-GFP(S65T)-hphMX6 的鉴定49
        3.3.3 菌株34-9的根癌农杆菌介导的转化49-50
        3.3.4 菌株 34-9 的醋酸锂转化50
        3.3.5 菌株 34-9 电转化方法50-51
        3.3.6 转化子的获得和初步鉴定51-52
        3.3.7 转化子遗传稳定性鉴定52-53
4 讨论53-57
    4.1 生防酵母菌遗传改良的可行性53
    4.2 原生质体融合53-55
        4.2.1 融合子筛选和鉴定53-54
        4.2.2 融合子筛选54
        4.2.3 融合子的检测54-55
    4.3 基因工程55
    4.4 电击转化的效率55-57
下一步研究计划57-58
参考文献58-69
在读期间发表的文章69-70
致谢70-71
附图及说明71-72
        


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