【作者】 王志成；

【导师】 易自力； 蒋建雄；

【作者基本信息】 湖南农业大学 ， 植物学， 2009， 博士

【摘要】 稻瘟病严重威胁着水稻生产,溶菌酶能够分解细菌或真菌细胞壁组分中多糖的糖苷键,从而抵御病原菌的侵染。因此利用溶菌酶基因转化籼稻,培育广谱高抗稻瘟病新种质,是水稻抗病育种的有效途径。本文以溶菌酶基因为目的基因,对农杆菌介导法和穗颈注射法转化超级杂交稻亲本不育系PA64s,恢复系R640、9311、MH63进行了系列研究,并对其转基因植株后代进行了遗传稳定性分析和抗性鉴定,将筛选出的优良抗病株系与其他亲本进行了杂交配组。主要研究结果概括如下:1优化了籼稻离体再生体系培养基以MS、NB和CC基本培养基中大量元素、微量元素Ⅰ（锰、锌、硼）、微量元素Ⅱ（铜、钼、钴、碘）、有机成分四种组分设计L₉（3⁴）正交试验,优选出了四个籼稻材料愈伤组织诱导、继代及分化最优的组分,获得了不同阶段的最佳培养基。研究结果表明,NB培养基中微量元素Ⅰ、NB或MS培养基中的有机成分有助于愈伤诱导,单独使用2,4-D愈伤诱导率高于不同浓度2,4-D和BA配合使用或BA单独使用;MS有机成分和大量元素、NB微量元素Ⅰ有助于愈伤增殖;NB大量元素和微量元素Ⅰ、10×CC微量元素Ⅱ有助于愈伤分化。采用优选培养基培养PA64s、9311、R640、MH63四个籼稻材料,平均愈伤诱导率为86.6%,继代25 d平均增殖倍数为3.85,平均分化率为88.1%。2优化了籼稻愈伤组织农杆菌转化方法对根癌农杆菌EHA105转化籼稻愈伤组织的技术体系进行了优化,其优化后的程序为:在农杆菌侵染液中添加终浓度为120μM的乙酰丁香酮,侵染液浓度为OD₆₀₀为0.4～0.6,侵染愈伤组织时间控制在20～30 min,-0.8MPa负压处理10 min,共培养时在共培养培养基表面覆盖一层滤纸,共培养3 d,侵染后的愈伤组织在抑菌培养基中恢复生长2周,先用150 mg/L的G418进行筛选,再用165 mg/L G418进行筛选。转化方法优化后G418筛选结果为:4个籼稻材料的平均抗性愈伤率为17.77%,平均抗性植株率为3.94%。3优化了穗茎注射法研究结果表明,穗颈节下第一节间部位注射,注射时期为主穗叶枕距7 cm以上,质粒浓度为500μg/ml,质粒DNA使用TE缓冲液溶解,R640、9311、MH63三个受体材料转化率皆达到7%以上,且三个品种的转化率差异不显著,说明优化的转化方法适合于不同的品种。进一步用携带了溶菌酶基因的工程农杆菌菌液进行穗茎注射,得到9株阳性株,转化率为0.03%。PCR检测初步确定目的基因与标记基因同时整合到基因组,Southern检测及后代分离比例证实为单拷贝整合。该方法以种质系统为受体,其整合方式可能是农杆菌整合方式,结合了农杆菌介导法和穗茎注射法的优势,是值得探索的一条转化途径。4获得了抗稻瘟病的转基因植株通过农杆菌介导法获得转化植株55株,通过质粒DNA穗茎注射法获得转化植株98株,通过农杆菌菌液穗茎注射法获得转化植株9株,经PCR和Southern杂交检测,证实了外源溶菌酶基因的整合。转化植株后代分离比的追踪分析结果表明,农杆菌介导法和农杆菌菌液穗茎注射法获得的转基因植株全为单拷贝整合,T1、T2代分离比基本符合孟德尔遗传规律。质粒DNA穗茎注射法获得的转化植株部分为单拷贝整合,部分植株为多拷贝整合。经过对转化植株T1、T2代的稻瘟病抗病性鉴定、PCR检测及纯合度检测,最终从2代中鉴定出21株“中抗”的阳性纯合转基因单株。5获得了抗稻瘟病的新杂交组合将筛选出的21个中抗纯合转基因株系作为亲本进行杂交配组,得到33个组合。抗性鉴定结果表明,与对照组合（抗性级别为“感”）相比,33个组合稻瘟病抗性皆有明显提高,3个组合表现为“抗”,26个组合表现为“中抗”,4个组合表现为“中感”;农艺性状考察结果表明,与对照组合相比,33个组合中22个组合农艺性状无显著差异,11个组合单株产量下降。综合抗性鉴定与农艺性状考察,获得了18个在生产上有应用前景的抗稻瘟病的新杂交组合,新杂交组合的进一步研究正在进行中。更多还原

【Abstract】 Rice blast is responsible for large yield loss in epidemically favorable areas and crop seasons.Lysozyme can decompose the cell wall of bacteria and fungi,resist the invasion of pathogens.Therefore,obtaining new germplasm with broad-spectrum blast-resistance by introduction of lysozyme gene into indica rice has been considered as an effective and economical strategy to control the disease.In this thesis,the Lysozyme gene was introduced into indica parents PA64s,R640, 9311 and Minghui63 by Agrobacterium-mediated,plasmid earstem injecting and Agrobacterium earstem injecting transformation systems.The factors influencing the transformation efficiency were investigated.Genetic stability analysis and blast-resistance identification of transgenic progenies were performed.And new hybridization combinations from the transgenic lines and other hybrid rice parents were selected.The main research contents and results were as follows:1 Optimization of culture medium of in vitro regeneration system of indica riceAnalyzing the organic maters,macro elements,micro elements-Ⅰ（manganese, zinc,boron） and micro elements-Ⅱ（copper,molybdenum,cobalt,iodine） of CC,NB and MS basic media,an orthogonal experiment of L₉（3⁴） was carried out to optimize the compose of media on calli induction,subculture and buds differentiation of indica rice.The result showed:Micro elements-Ⅰof NB,organic maters of NB or MS contributed to callus induction,and the calli induction ratio on the medium with 2,4-D alone was higher than that of the combination of 2,4-D and BA or BA alone.Organic maters and macro elements of MS,micro elements-Ⅰof NB contributed to the growth of callus.Macro elements and micro elements-Ⅰof NB acted significant role on differentiation,10 times of micro elements-Ⅱof CC was appropriated to differentiation. On the optimized media,the average calli induction ratio of 4 indica rice was 86.6%,the average calli subculture proliferation ratio after sub-cultured 25 d was 3.85, the adventitious buds induction ratio was 88.1%.2 Optimization of Agrobacterium-mediated transformation system for indica riceWhen OD₆₀₀ was 0.4-0.6,the inoculum with 120μM AS was very strong vitality. 20 min to 30 min of infection time,callus dealt with negative pressure of-0.8MPa for 10 min,co-cultured 2-4 d before screening,covered with a filter paper on the co-culture medium,were suitable for the infection.Increasing gradually the screening concentration of G418（150 mg/L at first,then 165 mg/L） after 2 weeks’ resumption culture can increase the resistant calli ratio.As a result,the average resistant callus rate of 4 varieties was 17.77%,and average resistant plant rate was 3.94%by G418 screening.3 Optimization of the earstem injection（1） Earstem injection with plasmidOn the development stage of mature embryo sacs,the distance between pulvinus longer than 7 cm,plasmid DNA of 500μg/ml dissolved with TE was injected into the first internode under the neck-panicle node.Transgenic lines were obtained,and the transformation rate of the three indica rice（R640,9311,MH63） was reached 7%. There was no significant differences on the transformation rate of the three indica rice, which showed that the optimized method was suitable to different indica varieties.（2） Earstem injection with AgrobacteriumNine transgenic lines were obtained by Agrobacterium earstem injection with lysozyme gene,the transformation ratio was only 0.03%.The transgenic lines were confirmed by NPTⅡgene and lysozyme gene PCR detection.The result of Southern blotting and the segregation ratios of progenies showed that the lysozyme gene was integrated as a single copy.With Agrobacterium integration mode,taking germ lines as receptor and combined with the advantages of Agrobacterium transformation system and earstem injection,this transformation system is a new transformation system worthy to be explored.4 Transgenic plants with blast resistance were obtained 55 transgenic plants were obtained by Agrobacterium transformation system,98 by plasmid earstem injecting and 9 by Agrobacterium earstem injecting.The lysozyme gene integrated into rice genome was confirmed by PCR and Southern blotting analysis.Segregation ratios analysis of transgenic progenies showed,the exogenous gene were integrated with sigle copy in the transgenic plants by Agrobacterium transformation and Agrobacterium earstem injecting,and the segregation ratios in T1 and T2 progenies were agreement with Mendelian genetic law expected from a single dominant gene segregating.But part of the transgenic plants by plasmid earstem injecting were confirmed integrated with single copy,part with multi-copy.21 moderate resistance,positive,homozygous T2 plants were selected by resistance identification,PCR detection and identification of homozygous of T1 and T2 plants.5 Superior hybridization combinations with good blast resistance were obtainedThe 21 homozygous lines were hybridized with corresponding parents. Identification of disease resistance showed,the blast-resistance was improved significantly in all the hybridization combinations compared with the controls （resistance degree were susceptible）,3 combinations showed resistance,26 combinations showed moderate resistance,4 combinations showed moderate susceptible.Investigation of agronomic traits showed,there was no significant differences among 22 combinations and the controls,the yield per plant of 11 hybridization combinations was significantly lower than that of the control.Combining the identification of disease resistance and investigation of agronomic traits,18 combinations with blast resistance were obtained,and further study of the 18 new combinations was in process.更多还原