Improving The Stress Resistance Of Rice By Exogenous MYB Gene Expression

MYB gene family is the largest transcription factor family in plant kingdom,and R2R3-MYB related genes belong to the largest subfamily of MYB gene.R2R3-MYB proteins have been repotorted to regulate developmental processes,primary and secondary metabolism of plant,also to be involved in the responses to biotic and abiotic stresses.Am ROSEA1,a R2R3-MYB proteins,is a key factor regulating the spatial distribution and intensity of pigmentation in both the corolla tubes and lobes of A.majus.Am Rosea1 has been introduced into several dicotyledonous species,such as tobacco,tomato,petunia,cotton,which gave rise to the accumulation of anthocyanin and other second metabolites significantly.In order to study the effect of Am Rosea1 ectopic expression on secondary metabolism and stress tolerance in rice,we introduced Am Rosea1 into rice(Oryza sativa L.),and examined the anthocyanin and flavonoid content,salt stress tolerance and drought tolerance of transgenic plants.We found that Am ROSEA1 could not effectively induce anthocyanin and flavonoid biosynthesis in rice,but clearly improved the tolerance of transgenic rice to drought and salinity stresses.We used Illumina sequencing technology to analyze the transcriptomes of wild type(WT)and transgenic rice plants under drought and salinity conditions and discovered that the transcriptional level of a considerable number of stress-related genes was affected by exogenous Am ROSEA1.These affected genes were involved in stress signal transduction,hormone signal pathway,ion balance and the removal of reactive oxygen species(ROS).The experimental results are as follows: 1、Generation of transgenic rice plants by overexpressing Am Rosea1We constructed a binary vector p CAMBIA1300-Am Rosea1,where Am Rosea1 c DNA was cloned under the 35 S promoter,and transformed it to rice(Oryza sativa L.).PCR analysis using Am Rosea1-specific primers confirmed the presence of the transgene in 24 T0 lines.By semi-quantitative RT-PCR,we screened transgenic lines OXR20,OXR21 and OXR22,in which the expression of Am Rosea1 was relative high.2、Measure anthocyanin and flavonoid contentTo test whether ectopic expression of Am Rosea1 in rice affected anthocyanin accumulation,using spectrophotometric method,we analyzed the content of anthocyanin and flavonoid in mature seeds and leaves at two developmental stages of rice,trefoil and heading.Results indicated that ectopic expression of Am Rosea1 did not change the content of anthocyanin and flavonoid in tested transgenic plants.3、Detect the tolerance of transgenic rice lines to high salt stress and drought stressAfter drought stress treatment,the survival rates of three tested transgenic lines OXR20,OXR21 and OXR22,were 78%±5.6%,85%±8.2% and 80%±7.1% respectively,all of which were higher than the 20.8%±1.7% survival rate of WT plants.After salt stress treatment,all WT plants died,whereas 70%±5.9% of OXR20,75%±5.6% of OXR21 and 69%±4.3% of OXR22 seedlings remained vigorous.These results showed that overexpression of Am Rosea1 enhanced tolerance to drought stress and high salt stress in transgenic rice lines.4、Transcriptome analysisIn order to investigate the molecular mechanism underlying the salt-tolerance and drought-tolerance of transgenic lines,transgenic line OXR21 was chosen for RNA-seq analysis as the representative of the ectopic expression lines.Analysis of differentially expressed genes(DEGs)that were responsible to stress showed that 64,282 and 50 genes were up-regulated,and 49,240 and 33 genes were down-regulated in the transgenic line compared to the WT line at 0,6 and 24 h of polyethylene glycol(PEG)treatment,respectively.In addition,49,197 and 58 genes were up-regulated,and 34,266 and 26 genes were down-regulated in the transgenic line compared to the WT line at 0,1 and 6 h of salt treatment,respectively.Venn analysis on DEGs revealed: eight and twelve genes were separately up-regulated at all the three time points of PEG treatment and all the three time points of salt treatment;twenty genes were separately up-regulated at both 6 h and 24 h of PEG treatment and both 1 h and 6 h of salt treatment;fifteen and twenty-six genes were separately up-regulated at 24 h of PEG treatment and 6 h of salt treatment.Gene Ontology(GO)analysis on DEGs revealed: within the category “biological processes”,six GO terms were enriched in the drought stress DEGs and salt stress DEGs,respectively.Four GO terms enriched in the sets of DEGs from the PEG group were also enriched in the salt group;they were “response to endogenous stimulus”,“response to abiotic stimulus”,“response to stress” and “response to stimulus”.KEGG pathway analysis revealed: the jasmonic acid signal transduction pathway was promoted and ethylene signal transduction pathway was repressed in transgenic plants compared to WT plants under both drought and salt stresses.Genes encoding high-affinity potassium transporter and fructosyltransferase that are related to osmotic protection and genes encoding catalases,peroxidases and ascorbate peroxidase that are related to protection from oxidative damage were differently expressed between WT and transgenic lines.In addition,stress responsible proteins,RD22,heat shock protein,LEA protein,RAB16A;transcriptional factors,DREB1 F,MYB48 and NAC134;signal transduction molecular serine-threonine kinase and CIPK14 were all differently expressed between WT and transgenic lines.In addition,the transcriptional level of some genes of unknown function was changed.In summary,Am ROSEA1 overexpression in rice rendered transgenic rice salinity and drought tolerance by direct or indirect regulation of diverse sets of genes.The endosperm of angiosperm seed originates from fertilized polar nuclei.The endosperm functions to nurture the embryo during the early stage of seed development.It also functions as a food reserve to nourish the growing seedling during germination in cereals with persistent endosperms.Moreover,cereal endosperms are the most important sources of food for global humans and livestock,and also provide raw materials for manufactured goods and biofuels.Therefore,studying the development of endosperm has both theoretical and economic benifits.Although great improvements have been achieved in the research on early endosperm development,little is known about PCD of endosperm.The b HLH transcription factor ZHOUPI encoded by At ZOU is a key regulator of endosperm PCD in Arabidopsis.As important grain crop and model species of monocotyledons,rice has persistant starchy endosperm.In order to study the function of ZOU homologous gene in rice endosperm development and to better understand the mechanism of rice endosperm degradation,we cloned the homologous gene of ZOU in rice,carried out gene expression analysis,functional complementation test,constructed GUS report vector,sense expression vector,antisense expression vector and transformed rice.The experimental results showed: 1、Evolution and expression analysis of ZOU gene in riceThere were two ZOU homologous genes in rice: LOC_Os04g35010(Os ZOU-1)and LOC_Os02g34320(Os ZOU-2).By real-time quantitative PCR,we detected their expression levels in root,stem,leaf of rice at vegetative growth stage,in glume of rice at jointing and booting stage,in seeds of rice in DAP2,DAP5,DAP7 and DAP9.It was found that Os ZOU-1 did not expressed in vegetative organs and in the seeds before fertilization,slightly expressed in DAP2 seeds,highest in DAP5 seeds,and the expression level in DAP7 and DAP9 seeds decreased gradually.Os ZOU-2 mainly expressed in stems and leaves,but not in seeds.2、Os ZOU-1 gene complements the phenotype of Arabidopsis zou-4 mutantThe phenotype of Arabidopsis zou-4 mutant transformed by At ZOUpromoter::Os ZOU-1 vector partly recovered,while the phenotype of Arabidopsis zou-4 mutant transformed by At ZOUpromoter::Os ZOU-2 vector was same with zou-4 mutant.3、The expression pattern of Os ZOU-1 in seed was detected by GUS stainingIn transgenic rice transformed by reporter vector Os ZOU-1promoter::GUS,GUS signal could not be found in DAP3 seeds,spread the whole endosperm of DAP5 seeds,and the signal area gradually narrowed to the dorsum of DAP7 and DAP9 seeds.4、The phenotype of transgenic rice plants with Os ZOU-1 constitutively expressingThe phenotype of transgenic rice plants transformed by vector Ubipromoter:: Os ZOU-1 was similar to Arabidopsis zou-1D mutant with At ZOU constitutively expressing: the plant was short,the leaves were narrow and curled up along the leaf edge,and the fertility was reduced.In addition,glumes cells were small,glumes were narrow and produced narrow seeds;starch endosperm accumulation was inadequate and could not completely fill the whole pericarp.5、The phenotype of transgenic rice plants with Os ZOU-1 silenced.The seeds of transgenic rice plants transformed by antisense vector ZOUpromoter:: Os ZOU-1 failed to start PCD with embryo adhereing to endosperm.Growth retardation of silenced plant seeds occurred at about eighth days after fertilization and seeds of silenced plant accumulated less starch than WT seeds.In summary,Os ZOU-1 regulates PCD of rice endosperm;seeds with Os ZOU-1 over-expressing or silenced accumulated less starch than WT seeds,which might imply that PCD affected starch accumulation.The innovations of this thesis:It is the first research to reveal that Am Rosea1 overexpression rendered transgenic rice salinity and drought tolerance by direct or indirect regulation of diverse sets of genes,which provides a possible way to utilize this gene to alter the abiotic tolerance in more rice cultivars.This study firstly explored the function of Os ZOU gene in regulating programmed cell death of rice endosperm,and the accumulation of starch in rice endosperm with abnormal PCD.This study helps us better understand the regulatory mechanism of programmed cell death and the relationship between nutrients metabolism and structure development in the process of rice seed development.