【作者】 邢莉萍；

【导师】 陈佩度；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 小麦白粉病是由白粉病菌（Ergsiphe graminis f.sp.tritici）引起的专性寄生真菌病害,在中国和世界小麦生产中危害日趋严重。由南京农业大学细胞遗传所发现的簇毛麦抗白粉病基因Pm21,对白粉病抗性强、抗谱广,已通过选育易位系途径导入普通小麦并被定位于6V染色体的短臂上。克隆Pm21基因并利用基因工程手段将其导入综合农艺性状优良的小麦品种中,对提高小麦白粉病抗性具有重要意义.本实验室在分离克隆抗白粉病基因研究中,已获得包括Pm21基因候选克隆在内的多个抗病相关基因。为了进一步鉴定这些抗病相关基因的抗性效应,本研究在优化小麦组织培养再生体系基础上,利用基因枪方法转化,将这些基因导入感病小麦品种,获得了一批转基因植株并对它们进行了分子检测和抗病性鉴定.为建立适于优良小麦品种遗传转化的高效组织培养体系,以20个优良小麦推广品种为试材,对以幼胚为外植体的愈伤诱导、分化和生根壮苗培养基进行筛选.试验结果表明,添加500mg/L水解酪蛋白、100mg/L脯氨酸、100mg/L谷氨酰胺,并以40g/L麦芽糖为碳源的MXD₂培养基的出愈率最高,愈伤质量最好.以大量元素减半的1/2 MX为基本分化培养基,添加1mg/L ZT和0.5mg/L IAA对愈伤组织诱导分化的效果最好,分化率最高达到83.0%.以1/2 MX培养基中添加0.5mg/L IAA和0.5mg/L MET的生根效果最好,且移栽成活率高.不同基因型的组织培养特性差异显著,本研究筛选到扬麦158、南农9918、济麦20、宁麦9号等8个基因型可作为优良的转基因受体材料,并初步建立起适合于它们的植株再生体系。同时为拓宽小麦组织培养外植体的范围,对扬麦158的成熟胚和幼穗的再生培养体系也进行了有益的探索。分别以扬麦158的幼胚、幼穗、成熟胚为外植体,在诱导培养基中添加不同激素组合,观察它们对愈伤组织诱导率、直接发芽率、分化率和愈伤组织质量、重量、生长速率的影响,建立起适合不同外植体的植株再生体系。本研究构建了包括小麦类甜蛋白基因Ta-Tlp、小麦Mlo反义基因、簇毛麦谷胱甘肽还原酶基因Hv-GR和Pm21基因的候选克隆簇毛麦丝氨酸/苏氨酸激酶基因Hv-S/TPK等多个抗白粉病相关基因的植物高效表达载体。在表达载体中均采用来源于玉米泛素基因的单子叶植物高效启动子ubi为目标基因的启动子,除草剂抗性基因bar为植物选择标记。通过基因枪将其导入到小麦品种扬麦158的幼胚愈伤组织中,采用上述优化的再生培养方案,在含bialaphos的选择培养基上经两轮筛选和分化,分别获得了这些基因的除草剂抗性再生植株。通过对T₀代再生植株进行PCR、Southern杂交与RT-PCR分析,获得转Ta-Tlp基因阳性植株27株,转小麦Mlo反义基因阳性植株45株,转Hv-GR阳性植株23株,转Hv-S/TPK阳性植株97株,平均转化率达到1.32%,转入的外源基因多数为多个拷贝。对T₀代及其衍生的T₁、T₂代转化植株进行白粉病抗性鉴定,结果表明,Ta-Tlp基因超量表达的转基因植株,能延缓白粉病发病进程,但其T₂代阳性转化植株的赤霉病抗性与受体对照无显著差异;部分转小麦Mlo反义基因的株系表现对白粉病中感到高抗;转Hv-GR基因植株的谷胱甘肽的氧化还原状态发生了改变,内源病程相关基因PR-la和PR-5被诱导表达,进而在一定程度上提高了转化植株对白粉病的抗性;值得注意的是转Hv-S/TPK基因的植株在整个生育期均表现高抗白粉病,叶片上仅有过敏性坏死斑和少量孢子堆出现。此外,还构建了包含Ta-LRR₂基因的的植物表达载体pBI-LRR₂,利用基因枪法将pBI-LRR₂和携带bar基因的表达载体pAHC25共转化,以高感白粉病的小麦品种扬麦158幼胚愈伤为受体,对转化再生植株进行PCR、Southern杂交与RT-PCR等分子检测,获得转Ta-LRR₂基因T₀代阳性植株89株。对T₀代和T₁转化植株进行白粉病人工接种的抗性鉴定,转化植株苗期多数叶片仅有过敏性坏死斑出现,表现为高抗白粉病;灌浆期大部分阳性植株虽有不同程度的发病,但仅在倒三叶以下及基部叶片,表现中抗白粉病.同时在T₁代筛选到了92株只有目标基因而无标记基因的转化植株,消除了标记基因带来的生物安全性疑虑,具有潜在的应用价值。更多还原

【Abstract】 Powdery mildew,caused by Erysiphe graminis f.sp tritici,is one of the most serious diseases of common wheat in China and many other countries in the world.A broadspectrum and powerful powdery mildew resistant gene,Pm21,was identified and introduced from H.villosa into common wheat through chromosome translocation,and located on the short arm of chromosome 6V in Cytogenetics Institute,Nanjing Agricultural University.The cloning of Pm21 will be critical for improving powdery mildew resistance level by genetic transformation.Several powdery mildew resistance-related and Pm21 candidate genes have been isolated from wheat or H.villosa.To characterize their potential functions by overexpression assays,it is necessary to establish a stable and high efficient transformation system.In the present research,the wheat tissue culture and regeneration system was first optimized.Immature embryo from twenty wheat cultivars was used as explant for tissue culture.Different hormone combinations and concentrations were applied for callus induction,differentiation and regeneration.The result showed that MXD₂ medium containing 500 mg/L Casein hydrolysate,100 mg/L Proline,100mg/L Glutamine and 40g/L Maltose was the best for callus inducion.As for callus differentiation,1/2MX medium containing 0.5mg/L IAA and 1mg/L ZT had the highest callus differentiation frequency of 83.0%.For plant regeneration,1/2MX containing 0.5mg/L IAA and 0.5mg/L MET was the best medium and obtained high frequency of surviving plant.Significant differences of response to tissue culture among wheat genotypes were observed.Eight varieties including Yangmai₁58,Nannong₉918,Jimai₂0,and Ningmai₉ et al.should be most suitable for immature embryo culture and regeneration and could be used for genetic transformation. Three different explant resources,immature embryo,mature embryo and inflorescence of Yangmai158,were further compared for their callus induction and plant regenerate ability to determine their potential use in wheat tissue culture.Callus induction mediums with different hormone concentration were used to study their effects on the frequency of callus induction,embryo germination,callus differentiation and quality,average weight,growth rate etc.Suitable plant regeneration culture system has been established.Five resistance-related genes,wheat thaumantin-like protein gene（Ta-Tlp）,wheat antisense Mlo gene（aTa-Mlo）,H.villosa glutathione geductase gene（Hv-GR） and H. villosa serine/threonine kinase gene（Hv-S/TPK）,were transformed by microprojectile-bombardment using powdery mildew susceptible wheat cultivars as receptor.Transgenic plants were identified by molecular genetic analysis and evaluated for their powdery mildew resistance.Four genes,Ta-Tlp,aTa-Mlo,Hv-GR and Hv-S/TPK,were constructed into plant expression vectors driven by the strong ubi promoter and the bar gene as selection marker.Plasmids were transformed into immature embryo-derived calli.After two rounds of herbicide bialaphos selection and regeneration,herbicide-resistant plants were identified.PCR,Southern blot,and RT-PCR analysis confirmed the presence of the transgenes in the host genome.Totally 21,45,23 and 97 transgenic plants for the above four genes were obtained respectively.Average transformation efficiency was as high as 1.32%.Most transformants had more than one trangene copy.The transgenic plants of T₀, T₁,and T₂ generations were evaluated for their disease resistance.The Ta-Tlp transgenic plants of T₀,T₁ and T₂ generations showed delayed disease development of powdery mildew,while no distinct improved scab resistance was observed compared with the susceptible receptor.Partial overexpressing antisense Ta-Mlo transplants showed moderate susceptible or high resistance of powdery mildew.Overexpression of Hv-GR enhanced the resistance to powdery mildew and induced transcript accumulation of other pathogenesis-related genes such as PR-1a and PR-5 through changing the redox state of inner glutathione. Overexpression of Hv-S/TPK transplants showed high powdery mildew resistance throughout the growing period,and produce only a few hypersensitive response spots and sporadic piles of spore on the leaves.Ta-LRR₂ gene was transformed into wheat using a co-transformation method,in which target gene Ta-LRR₂ and the bar gene were in separated vectors（pBI-LRR₂ and pAHC25, repectively） and co-transformed simutaneously.Eighty-nine Ta-LRR₂ transgenic plants were identified by PCR,Southern blot,and RT-PCR analysis.Average transgenic efficiency was about 1.85%.The transgenic plants of T₀ and T₁ generations were evaluated for powdery mildew resistance.All transgenic plants showed high powdery mildew resistance at the seedling stage,while some hypersensitive response spots were observed on the leaves. At the adult stage,the transplants showed moderate powdery mildew resistance,and the disease symptom was found on the bottom leaves of the plants.Ninty-two transplants,in which contained the target gene but no marker gene,were furthermore identified.This could escape the biosafety problems by selection marker gene and should be more useful in wheat improvement for disease resistance.更多还原