【作者】 李平；

【导师】 何光华；

【作者基本信息】 西南大学 ， 作物遗传育种， 2008， 博士

【摘要】 水稻是全球最重要的粮食作物之一,世界上半数以上的人以稻米为主要的粮食来源,其中大约90%的水稻被亚洲人生产和使用。水稻纹枯病和稻瘟病是水稻的两大主要病害,水稻纹枯病是由病原菌Rhizoctonia solani引起的,稻瘟病是由病原菌Magnaporthe grisea引起的。Lamp（1994）指出,每年增加的人口至少需要消耗0.13亿吨水稻,相当于全世界在1990到2025年间每年须有1.7%的水稻增产。Baker et al.（1997）研究表明在1975至1990年,因为稻瘟病大约有15.7亿吨水稻损失,几乎相当于全球水稻产量的11%-30%,而源于纹枯病的水稻损失大约在8-50%（Savary and Mew 1996）。因此,培育抗病水稻品种具有重要的现实意义。稻属抗性种质资源的匮乏、生态环境的变化及常规育种方法的局限性给水稻进一步遗传改良带来一定的困难。由于稻瘟菌的致病性分化严重,生理小种多,变化大,水稻自身抗性资源有限,再加上常规育种方法耗时费力,所培育的抗病品种远不能满足生产上的需要。对纹枯病来说,至今还没有发现高抗纹枯病的抗源,缺少抗纹枯病的抗性基因,因而导致水稻的纹枯病抗病育种进展更加缓慢。应用基因工程手段将异源具有广谱抗性的基因转入水稻,获得抗病性增强的转基因植株,实现了传统育种方法无法实现的基因重组分子植物育种,可以大大提高育种水平。本论文首先构建了籼稻缙恢35的农杆菌介导的遗传转化体系,然后利用酵母表达系统表达了来自苜蓿的防御素alfAFP基因,体外研究了表达蛋白对水稻病原菌的抑制影响。考虑到将具有不同功能的抗菌基因一起转入水稻可以提高转基因水稻的持久抗病性,所以构建来自苦瓜的几丁质酶McCHIT1、alfAFP及其双价基因的表达载体,转入水稻,获得转基因植株,并研究了其抗病性和后代农艺性状变化,主要的研究结果如下:一.籼稻农杆菌介导的遗传转化体系的建立1.表达载体的构建构建了McCHIT1、alfAFP单价基因和McCHIT1/alfAFP双价基因的表达载体。该载体包含了除草剂的筛选标记基因和一个GUS基因的报告基因,目的基因以玉米来源的Ubiqutin启动子启动表达。2.缙恢35遗传体系的建立建立了籼稻（缙恢35）的农杆菌介导的遗传转化体系。通过探讨和优化缙恢35的诱导、继代和分化再生的培养基成分、激素配比以及对农杆菌介导的遗传转化所涉及的某些参数,最后确定了缙恢35农杆菌介导的转化程序。获得了大量的再生植株:拟McCHIT1转基因植株495株,拟alfAFP转基因植株620株和拟McCHIT1/alfAFP双价转基因植株285株。3.转基因植株的初步检测通过对转化获得的再生植株进行GUS组织化学检测,结果表明总共有8.64%的阳性植株率。分别获得45株McCHIT1转基因植株、56株alfAFP转基因植株和20株McCHIT1/alfAFP双价转基因植株。通过目的基因的PCR初步检测,GUS阳性植株均扩增出了目的基因片段,说明外源基因已经转入水稻。二.alfAFP基因体外表达蛋白对水稻三大病原菌的抑制作用将alfAFP基因在毕赤酵母中进行分泌表达,其表达产物经过15%SDS-PAGE检测,其分子量大约为6.5 kDa。alfAFP重组蛋白在体外对水稻纹枯病、稻瘟病和白叶枯病菌有一定的抑制效果。在添加alfAFP酵母表达产物22.00μg时对纹枯病有较强的抑菌圈,在含有重组蛋白浓度为5μg/mL、10μg/mL、15μg/mL和20μg/mL的培养基上,50 h内对纹枯病菌丝的生长也有较明显的抑制效果。除此,在添加alfAFP酵母表达产物22.00μg时还分别对稻瘟病和白叶枯病原菌具有一定的抑制作用。三.McCHIT1、alfAFP及其双价转基因水稻的抗病性检测1.转基因水稻T₁代分离比检测通过对各转基因植株T₁代植株的GUS组织化学检测,GUS基因在T₁代分离比例多数符合孟德尔3:1的分离比,表明外源基因多以单拷贝的形式整合转入水稻中。2.各转基因水稻的纹枯病抗性检测各转基因植株多世代（T₀、T₁和T₂代）抗纹枯病检测结果表明,McCHIT1、alfAFP单基因和McCHIT1/alfAFP双价转基因植株均显著提高了对纹枯病的抗性,病情指数较对照植株均下降,其中McCHIT1转基因植株11株,alfAFP转基因植株11株以及McCHIT1/alfAFP双价转基因植株6株。以T₂代植株抗性检测为例,各转基因植株病情指数较对照相比下降幅度分别为28.42-42.86、28.24-53.63、25.74-43.96。3.各转基因水稻对稻瘟病的抗性检测各转基因植株多世代（T₀、T₁和T₂代）的稻瘟病检测结果表明,McCHIT1、alfAFP单基因和McCHIT1/alfAFP双价转基因植株均显著提高了对稻瘟病的抗性,病情指数较对照植株均下降,其中McCHIT1转基因植株12株,alfAFP转基因植株11株以及McCHIT1/alfAFP双价转基因植株5株。以T₂代植株抗性检测为例,各转基因植株病情指数较对照相比下降幅度分别为31.57-85.73、27.92-57.48、37.81-56.47。此外各转基因植株还表现出对来源不同的多种稻瘟菌的抑制作用。4.转基因水稻抗病性的比较对McCHIT1/alfAFP转基因水稻抗病性比较分析,结果表明,McCHIT1基因在提高转基因水稻对纹枯病的抗性方面表现相对好一些,而alfAFP基因在提高转基因水稻对稻瘟病的抗性方面表现相对好一些。McCHIT1/alfAFP双价转基因植株虽然一定程度提高了对纹枯病和稻瘟病的抗性,但是没有发现两个基因明显的协同增强作用。5.转基因表达水平对抗病性的影响通过RT-PCR分析了目的基因在转基因水稻中的表达水平,并评价了和抗性水平之间的关系。实验结果表明,McCHIT1和alfAFP基因在转基因植株中的转录水平上均得到有效表达,并且不同的转基因植株的表达水平有所差异。在双价转基因植株中,两个基因的表达水平表现不一致。在一定条件下,具有更高水平的目的基因表达植株通常具有更高的抗病水平,但也有极少数转基因植株虽然具有较高目的基因的表达水平却没有表现出相应的抗病水平。四.转基因植株后代主要农艺性状分析分析所有测试转基因植株T₂代的8个主要农艺性状,结果显示,与对照植株相比,大多数转基因植株后代的8个农艺性状均有不同程度的变异,总体上在T₂代出现了植株矮小或者明显高大,育性降低,分蘖力降低,穗长、穗粒数、千粒重明显下降等变异。且某些测试的转基因植株在某些性状表现更优良。1.McCHIT1转基因植株的农艺性状分析调查的8个主要农艺性状测定结果表明,株系间各农艺性状存在一定程度的差异。在各转基因株系中,株高、有效穗、穗长、单穗总粒数、结实率、千粒重、单穗实粒重和单株实粒重的变异幅度分别为76.2-125.9 cm、4.6-20.2穗、24.8-29.4cm、184.0-294.5粒、14.3-82.6%、26.3-39.7 g、0.7-6.6 g和6.9-83.8 g。2.alfAFP转基因植株的农艺性状分析调查的8个主要农艺性状测定结果表明,株系间各农艺性状存在一定程度的差异。在各转基因株系中,株高、有效穗、穗长、单穗总粒数、结实率、千粒重、单穗实粒重和单株实粒重的变异幅度分别为88.1-115.0 cm、6.6-13.6穗、25.9-27.6cm、162.4-219.8粒、52.7-87.1%、26.1-35.7 g、3.0-5.1 g和20.9-68.8 g。3.McCHIT1/alfAFP转基因植株的农艺性状分析调查的8个主要农艺性状测定结果表明,株系间各农艺性状存在一定程度的差异。在各转基因株系中,株高、有效穗、穗长、单穗总粒数、结实率、千粒重、单穗实粒重和单株实粒重的变异幅度分别为78.3-111.0 cm、5.4-13.4穗、23.6-26.7cm、155.1-219.5粒、37.6-79.3%、25.6-31.8 g、1.8-4.8 g和20.6-55.1 g。4.各转基因植株农艺性状的变异系数与相关性在不同基因的转基因植株中,株高、穗长、每穗总粒数和千粒重性状变异系数较小,而有效穗、结实率、穗实粒重和株实粒重性状的变异系数较大。与单株实粒重成显著正相关的性状在不同基因的转基因植株中存在一定差异,且具有不同大小的相关系数。某些具有负相关的性状在不同基因的转基因植株中也具有一定的差异。更多还原

【Abstract】 Rice（Oryza sativa L.） is one of the world’s agronomically important cereal crops, providing food for more than a half of the global population.About 90%of the world rice is produced and consumed in Asia.Rice blast and sheath blight,seriously affecting yield and quality of rice worldwide,are caused by fungal pathogens Magnaporthe grisea and Rhizoctonia solani respectively.It is predicted that an additional 13 million tons of rice per year will be required to keep up with the rapid growth in rice consuming populations.This translated to an annual increase of 1.7%in world rice production between 1990 and 2025（Lamp 1994）.And one hundred and fifty seven million tons of rice has been lost to rice blast disease from 1975 to 1990,a figure that equals 11 to 30% of global rice production（Baker et al.1997）.The yield losses have ranged from 8 to 50%due to sheath blight,particularly when the infection is well distributed and severe in the field（Savary and Mew 1996）.Therefore,it is significantly important to cultivate disease-toleranted rice cultivar.However,due to the deficiency of rice resistance germplasm resources,the changes of ecological environment and the limitations of conventional breeding,it causes some difficulties for further rice genetic improvements and has been of limited scope in controlling the pathogen.For Magnaporthe grisea,the cultivation of resistant varieties is far from meeting the production requirements,because of serious differentiation of its pathogenicity,the appearance of new physiological races,the deficiency of rice’s own resistance genes,let alone the time-consuming of traditional breeding.For sheath blight resistance,so far,only one locus has been mapped on chromosome 5 of rice.With the development of modern biotechnology,genetic engineering is a powerful tool to transform exogenous genes for molecular breeding of rice cultivars resistant to these two fungi.In this study,firstly,we described an improved protocol for Agrobacterium-mediated transformation of indica（JinHui35） rice.Secondly,the alfAFP gene was expressed in yeast（Pichia pastoris） and assessed antimicrobial activity against rice main pathogens in vitro.Thirdly,in order to obtain durable disease-resistaned of rice,the different functional genes were used.The expression vectors of McCHIT1,alfAFP and McCHIT1/alfAFP bivalent gene were constructed and transformed into rice mediated by Agrobacterium tumefaciens.Some transformants were obtained.Disease tolerance against rice three main pathogens of transgenes were evaluated in planta inoculation assays,and the main agronomic traits of transgenic lines were also analyzed.The important results were as follows:1.The establishment of indica genetic transformation system mediated by Agrobacterium tumefaciens（1）.The construction of expression vectorsThe expression vector of McCHIT1,alfAFP and McCHIT1/alfAFP bivalent genes driven by ubiquitin promoter from maize were constructed.The vector also harbored expression cassettes of the Bar gene as selectable marker and theβ-glucuronidase（GUS） gene as transformation reporter.Then,the expression vector was transformed into A. tumefaciens strain EHA105 by electroporation to obtain engineering strains.（2）.The establishment of genetic transformation system of JinHui35To establish the Agrobacterium-mediated genetic transformation system,some important factors influencing genetic transformation of indica rice were studied, including the induction and subculture of calli from JinHui35,the type and compositions of medium,the combination of different hormones influenced the regeneration frequency,the key factors influencing Agrobacterium-mediate genetic transformation.Finally,495 putative McCHIT1 transgenic plants,620 putative alfAFP transgenic plans and 285 putative McCHIT1/alfAFP transgenic plants were obtained.（3）.The preliminary analysis of transgenic plantsThe results showed the ratio of GUS-positive plants was 8.64%by GUS histochemical examination analysis.45 McCHIT1 transgenic plants,56 alfAFP transgenic plans and 20 McCHIT1/alfAFP transgenic plants were obtained.PCR assay exhibited primarily that foreign genes had already been integrated into rice.2.Secreted expression of alfAFP in Pichia pastoris and its antimicrobial activity against rice pathogens in vitroIn this research,alfAFP gene was inserted into yeast expression vector pPIC9K, and then transferred into Pichia pastoris GS115 strain by electroporation.The alfAFP gene was expressed in yeast and utilized to assess antimicrobial activity in vitro.A molecular mass of expressed alfAFP was about 6.5 kDa by 15%SDS-PAGE analysis, and the recombinant protein showed certain antimicrobial activity against rice three main pathogens.22.00μg alfAFP showed antimicrobial activity against Rhizoctonia solani,Magnaporthe grisea and Xanthomonas oryzae pv.Oryzae.Moreover,5μg/mL、10μg/mL、15μg/mL and 20μg/mL alfAFP protein also showed inhibitation of R. solani no more than 50 h. 3.Enhanced resistance to rice pathogens in transgenic rice overexpressing McCHIT1,alfAFP and McCHIT1/alfAFP（1）.Analysis of segregation ratio of transgenic lines in T₁ generationMost T₀ transformants were consistent with 3:1 segregation ratio by x² test according to GUS staining in T₁ generation,which showed that most transgenes were inherited as a single-copy Mendelian trait.（2）.Enhanced resistance to R.solani in transgenic riceTransgenic rice overexpressing McCHIT1,alfAFP and McCHIT1/alfAFP were inoculated by R.solani in T₀,T₁ and T₂ generations.The results showed the expression of McCHIT1,alfAFP and McCHIT1/alfAFP in transgenic rice enhanced the resistance to R.solani,relatively,which contained 11 McCHIT1-transgenic lines,11 alfAFP-transgenic lines and 6 McCHIT1/alfAFP-transgenic lines.In bioassay of T₂ transgenic plants,for example,at 14 d after inoculation of R.solani,control plants were regarded as 100,the disease indices of tested McCHIT1,alfAFP and McCHIT1/alfAFP-transgenic lines were 28.42-42.86,28.24-53.63,25.74-43.96 decrease.（3）.Enhanced resistance to M.grisea in transgenic riceTransgenic rice overexpressing McCHIT1,alfAFP and McCHIT1/alfAFP were inoculated by M.grisea in T₀,T₁ and T₂ generations.The results showed the expression of McCHIT1,alfAFP and McCHIT1/alfAFP in transgenic rice enhanced the resistance to M.grisea,relatively,which contained 12 McCHIT1-transgenic lines,11 alfAFP-transgenic lines and 5 McCHIT1/alfAFP-transgenic lines.In bioassay of T₂ transgenic plants,for example,at 10 d after inoculation of M.grisea,control plants were regarded as 100,the disease indices of tested McCHIT1,alfAFP and McCHIT1/alfAFP-transgenic lines were 31.57-85.73,27.92-57.48,37.81-56.47 decrease.Moreover,different transgenic plants were also exhibited non-specific resistance to different M.grisea strains.（4）.Disease resistance from McCHIT1/alfAFP transgenes against different pathogensThe resistance of the McCHIT1/alfAFP transgenic plants to different pathogens was determined.The results showed that McCHIT1 gene in transgenic rice exhibited higher resistance to R.solani,and alfAFP gene performed higher resistance to M.grisea, according to the decrease of disease indices compared to control plants.Although McCHIT1/alfAFP-transgenic plants enhanced resistance to sheath blight and rice blast, the bivalent transgenic plants didn’t showed significant synergistic action of these two genes to rice pathogens.（5） Influence of exogenous genes expression level to the disease resistance in transgenic plantsThe relative transcription levels of McCHIT1 and alfAFP in transgenic plants were determined by RT-PCR analysis.The results showed that differences of McCHIT1 and alfAFP expression levels existed among various transgenic lines.This two gene expression levels were usually different in bivalent transgenic plants.Generally speaking,individual transgenic plants showing higher gene expression levels always exhibited higher disease resistance levels with a certain range.But there are a few of transgenic plants with high levels of gene expression did not show the appropriate level of disease.4.Analysis of the main agronomic traits of T₂ transgenesThe 8 main agronomic traits of all tested transgenic plants were analyzed,most transgenic plants showed variation among different agronomic traits to some extent, respectively,as compared with control plants.On the whole,as compared with control plants,most transgens showed certain different variation with shorter or taller plant height,lower fertility,less effective panicles,less grains per panicle and less 1000-grains weight,etc.And even some transgenes showed better chatacters.（1）.Analysis of the main agronomic traits of T₂ McCHIT1-transgenic plantsThe range of variation in plant height,effective panicles,panicle length,grains per panicle,seed setting rate,1000-grains weight,yield per panicle and yield per plant in T₂ McCHIT1-transgenic plants were 76.2-125.9 cm,4.6-20.2,24.8-29.4 cm,184.0-294.5, 14.3-82.6%,26.3-39.7 g,0.7-6.6 g,6.9-83.8 g.（2）.Analysis of the main agronomic traits of T₂ alfAFP-transgenic plantsThe range of variation in plant height,effective panicles,panicle length,grains per panicle,seed setting rate,1000-grains weight,yield per panicle and yield per plant in T₂ alfAFP-transgenic plants were 88.1-115.0 cm,6.6-13.6,25.9-27.6 cm,162.4-219.8, 52.7-87.1%,26.1-35.7 g,3.0-5.1 g,20.9-68.8 g.（3）.Analysis of the main agronomic traits of T₂ McCHIT1/alfAFP-transgenic plantsThe range of variation in plant height,effective panicles,panicle length,grains per panicle,seed setting rate,1000-grains weight,yield per panicle and yield per plant in T₂ McCHIT1/alfAFP-transgenic plants were 78.3-111.0 cm,5.4-13.4,23.6-26.7 cm, 155.1-219.5,37.6-79.3%,25.6-31.8 g,1.8-4.8 g,20.6-55.1 g.（4）.The variation coefficient and correlation coefficient of agronomy traits in different transgenesThere was less variation in plant height,panicle length,grains per panicle and 1000-grains weight,and more variation in effective panicles,seed setting rate,yield per panicle and yield per plant in different transgenic lines（McCHIT1,alfAFP and McCHIT1/alfAFP-transgenic lines）.On the other hand,there was different correlation coefficient of 8 agronomy traits in different transgenes,moreover,the traits which were significantly positive correlation with yield per plant and the traits with tenuous negative correlation among different transgenes were also different.Results from this study will serve as a foundation for further investigations into rice defense mechanisms and provide the new transgenic rice varieties with enhanced resistance against the rice pathogens.更多还原