大丽轮枝菌致病性相关突变体快速筛选体系的建立(2)
时间:2015-10-14 14:52
来源:发表吧
作者:王新艳，张丹丹，桂月
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鉴于此，本研究在传统方法的基础上，明确了大丽轮枝菌孢子浓度与棉花黄萎病发病程度的关系，确定引起棉花黄萎病的孢子悬浮液浓度范围为大于5.0×105孢子/mL（图1），为突变体孢子悬浮液的快速定量制备提供了理论依据；进一步通过标准化培养大丽轮枝菌和孢子悬浮液洗脱方法，确定定性制备孢子悬浮液的洗脱体积为25mL，批量测试的孢子浓度均满足接种要求（图2），解决了接种时需要逐一定量测定突变体孢子浓度的问题。接种方面，采用了棉花幼苗裸根浸泡于孢子悬浮液的方式，接种均一，发病周期短（仅需14d），解决了根系接种不均一、发病周期长的问题。同时，建立的标准化方法每个突变体仅接种5株棉花幼苗，结果统计简单易行，解决了接种要求苗数多、占用资源多的问题。由此建立了大丽轮枝菌致病性相关突变快速筛选体系（图3），该方法首选分别取20μL保存于96孔板的甘油菌液于培养皿中涂布培养5d以分离单孢，随后挑取单孢于培养瓶中培养9d，加入25mL的灭菌水洗脱孢子，并将准备好5株棉花幼苗置于培养瓶中处理，接种后置于营养钵中继续培养14d，调查统计结果。在此基础上，统筹棉花幼苗种植、突变体单孢分离、突变体培养、接种和结果统计等环节，建立了大丽轮枝菌致病性快速筛选体系。该体系是以保存于96孔板的突变体为整体单位，规范了突变体培养、孢子悬浮液制备、接种、继续培养和调查统计流程，通过各个环节突变体一一对应操作和时间节点统筹设计，实现了大规模突变体的流水线鉴定。应用该体系，1个循环7个流程可以完成1344个突变体的筛选鉴定（图4），鉴定周期为54d，其中工作量仅为21人日，提高筛选效率和减少工作量均在10倍以上。按笔者实验室16000个突变体库计算[26]，两名科研人员一年内即可完成突变体库中致病性相关突变体的筛选。
建立的筛选体系尽管解决了致病性相关突变体快速筛选的问题，但由于接种苗数较少，可能出现部分假阳性，这是由于在接种均一的前提下，棉花幼苗个体间对病原菌敏感性不一致和少量突变体悬浮液孢子浓度偏低（产孢能力受限的突变体）造成的。解决方法是将筛选出靶标突变体再次鉴定，以本研究鉴定的致病性相关突变体的比例（1344个突变体中69个致病力显著下降）测算，16000个突变体约有800多个致病力相关突变体，仅需不到1个循环即可获得重复验证结果，提高筛选的准确性；或者采用定量菌液法验证致病下相关突变体的可靠性，如蛭石沙土无底纸钵定量蘸菌液法[24]。同时，应用本筛选体系还需注意两个问题：一是筛选致病力下降不明显的突变体准确性偏差，建议对获得的靶标突变体进行二次筛选验证；二是对于致病力增强的突变体需要通过观察发病进程的来判断，且最好选用耐或者抗病的品种作为鉴定材料。
4结论
明确了引起棉花黄萎病的大丽轮枝菌孢子悬浮液浓度为大于5.0×105孢子/mL，建立了大丽轮枝菌的标准化培养和孢子悬液制备方法，甘油菌种活化培养5d，标准培养瓶扩大培养9d，确定了25mL灭菌水为适宜的孢子洗脱体积，定量制备的孢子悬浮液适合于接种棉花幼苗并引起黄萎病。在此基础上，建立了大丽轮枝菌致病相关突变体的快速筛选体系。应用该体系测试表明，1人1个循环7个流程可完成1344个突变体的快速筛选体系，鉴定周期仅54d，工作量仅为21人日，极大地缩短了筛选周期。同时，测试获得69个致病力显著下降的突变体，对筛选到的致病性相关突变体进行定量菌液接种验证，结果一致。研究建立的快速筛选体系适用于T-DNA随机插入突变体库的筛选鉴定，这将为后续大丽轮枝菌致病性相关突变体的批量筛选提供保障。
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