【作者】 张立；

【导师】 韩德俊；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2012， 硕士

【摘要】 小麦是我国主要的粮食作物，其产量需求随着经济水平的提高和人口的快速增长而提高。小麦生产中的各种病害已成为制约其产量和品质提高的重要因素，然而常规育种在改良品种抗性中具有诸多不足，难以满足经济社会发展的要求；生物技术育种的发展给小麦抗病育种提供了更广的可利用基因资源，同时具有更短的育种周期和更广泛的特性改良，已成为抗病育种中的重要补充手段。良好的外源基因是生物技术育种的重要前提，本研究所采用的紫花苜蓿抗真菌肽alfAFP基因是来自苜蓿种子的防御素基因，转基因马铃薯表现出对主要病原真菌Verticillium dahliae的优良抗性。另一外源基因spCEMA是在具有抑菌活性的阳离子抗菌肽CEMA基础上添加信号肽改造而来的。本研究主要使用这两类基因，针对小麦生产品种进行转化操作，采用的方式以基因枪法为主，同时采用了农杆菌介导法和花粉管通道法，并对各转化操作中的若干问题进行了研究，以期建立稳定高效的小麦转化体系并获得转化植株。1基因枪介导融合基因转化小麦本研究采用紫花苜蓿抗真菌肽alfAFP基因和抗菌肽spCEMA基因作为目的基因，通过Overlapping PCR构建成由连接肽2A序列连接的融合基因alfAFP:2A:spCEMA并连接到植物表达载体pWMB002和pRTL2上，与含bar基因的标记基因载体pAHC20共转化西农1376，获得再生植株，并对bar基因和融合基因alfAFP:2A:spCEMA进行PCR检测。获得同时含有目的基因及bar基因的双转化植株7株，共转化率为0.1％；转基因植株共61株，总体转化率为0.86％。对共转化操作及筛选过程中影响再生率和转化率的若干因素进行了研究，建立了针对西农1376的共转化体系。2农杆菌介导法转化小麦利用不同的农杆菌菌株转化小麦的茎尖生长点、未分化幼穗、开花期花器官和愈伤组织，获得了一系列不同的再生结果。GUS组织化学染色显示了瞬时表达的存在，但由于筛选方式导致的生根问题，并未获得再生成株植株。3花粉管通道法转化小麦利用构建的植物表达载体转化人工授粉后去柱头的小麦品种西农1376、小偃22和小偃6号，导入外源目的基因，并利用bar基因作为标记基因检测转化后代。对目的基因alfAFP的后代通过连续喷涂Basta，在T1代获得了转基因再生植株，并通过PCR检测，该处理流程bar基因筛选效率为5％。本研究通过基因枪共转化法、农杆菌介导法和花粉管通道法系统研究了小麦生产品种的转化问题，获得了转基因植株并对转化过程中的若干影响因素进行了探讨，为完善各转化方式提供了参考。更多还原

【Abstract】 Wheat is the main grain in China, and its yield needs to be improved along with theeconomic development and the rapid population growth. Wheat disease is one of the mostserious factors affecting both the yield and the quality of wheat. As conventional breeding hasseveral deficiencies in meeting the requirements of economic and social development,biotechnology breeding becomes an important supplementary means of breeding for wheatdisease resistance, with more widely available gene resource and shorter time cycle comparedwith the general means.The exogenous gene is an important prerequisite for biotechnology breeding. The alfalfaantifungal peptide gene alfAFP used in this study is a plant defensin gene isolated from theseeds of Medicago sativa and prior studies have already demonstrated that it could displaystrong activity against the agronomically important fungal pathogen Verticillium dahliae intransgenic potato. The other gene used in this research is an antimicrobial peptide genespCEMA which is modified from CEMA, a cation antimicrobial peptide with highantimicrobial activity. These two genes are used in this wheat transformation study usingmainly biolistics, as well as the Agrobacterium-mediated transformation and the pollen-tubepathway. Serial problems were studied in order to construct a stable and efficient wheattransformation system and obtain transformed plants.1. Transformed wheat with fusion gene via biolisticsIn this study, the alfalfa antifungal peptide gene alfAFP and the antimicrobial peptidegene spCEMA were linked by the linker peptide2A sequence via Overlapping PCR and thefusion gene alfAFP:2A:spCEMA connected to the plant expression vectors pWMB002andpRTL2was used as the target gene in the following co-transformation process with pAHC20containing bar as a selective marker. Regenerated plants were obtained and PCR array wasdone to detect both bar and the target gene alfAFP:2A:spCEMA.In all,7transformed plants containing both bar and the target gene alfAFP:2A:spCEMAwere obtained and the frequency of co-integration was0.1%; a total61transformed plantswere regenerated and the overall transformation frequency was0.86%. Several transformationand regeneration related factors were studied during the screening and regenerated processand the co-transformation system of wheat cultivar XiNong1376was established.2. Agrobacterium-mediated transformation of wheat Different Agrobacterium strains were used to transform various portions of wheatincluding shoot tip, undifferentiated spike, floral organs as well as callus. Serial differentresults were obtained and transient expression were demonstrated via GUS histochemicalstaining. However no adult plants were regenerated due to the root developing problemcaused by the inappropriate screening methods.3. Pollen-tube pathway transformation of wheatWheat cultivars XiNong1376, XiaoYan22and XiaoYan6were transformed afterartificial pollination and cutting off the stigma and bar gene was used as the marker gene.Transformed offspring in T1generation of the regenerated plants with alfAFP as the targetgene were obtained after continuous spraying Basta and PCR array proved this screeningprocess is effective with a5%frequency of the detection of bar gene.In all, wheat transformation using biolistic co-transformation, Agrobacterium mediatedtransformation as well as the pollen tube pathway was done and transgenic wheat plantletswere obtained. Several influential factors of these conducting process were discussed in orderto improve each method.更多还原