Enhanced Resistance To Blast (Magnaporthe Grisea) And Sheath Blight (Rhizoctonia Solani) In Transgenic Indica Rice By Constitutive Expression Of Chitinase And Defensin Gene

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.