Molecular Mapping And Cloning Of A Narrow Leaf Gene Nal11/OsDnajc1

Rice is one of the most widely grown food crops and feed more than 1/3population in the world,it also has been the most ideal model plant for the study of growth and development of the single leaf plants.After two times of the green revolution,rice yield has been greatly improved.However,after entering twenty-first century,the world population continues to expand,soil erosion and degeneration caused decrease of arable land,sustained tension of agricultural water resources in many areas,global warming and the future demand for bio-energy has made the food crisis become more and more emergent.In order to improve rice yield,plant type breeding combined with heterosis and utilization of favorable geneshas become a consensus strategy to breakthrough the bottleneck of high yield breeding.As an important food crop,rice leaves are not only the main vegetative organs,but also play an important role in plant type and yield.The shape and size of rice leaves are closely associated with photosynthetic efficiency.Excellent plant morphology has high utilization rate of light energy,which is the basis of high yield.Through the improvement of rice leaf morphology to cultivate new varieties with ideal plant type has become a hot research topic.With the development of molecular biology technology,more and more genes associated with leaf morphology were identified.Localization and cloning of these genes for rice leaf has not only laid a theoretical foundation of leaf morphogenesis regulation of network,but also provide a practical basis for ideal type high yield rice breeding.Therefore,the study on leaf of rice is not only lay foundationof great theoretical significance,but also has high application value.In this study,we isolated a new rice narrow leaf genenal11（narrow leaf 11）/Osdnajc1by positional cloning.Thereafter,by morphology,physiology and molecular biology method,we conducted a preliminary analysis on the genetics,gene structure and expression pattern of this gene,the main results are as follows:1.Morphological and physiological analysisZYX is anindica riceshows serval special phenotypic traits,such as small panicle,narrow leaves,thin and slender stem,more tillers,low seed setting rate,semi-dwarf and so on.Through observation of paraffin sections,we found that,compared to 02428,the main reason of ZYX’s narrow leaf wasdue to the decrease in the number of small veins,and reduced number of small vascular bundles result inthin and slender stem;by using scanning electron microscope,compared with 02428,stomatal number per mm²is smaller,number of papilla cells aroundstomatal is also reduced;the content of endogenous hormones in rice seedling stage leave was detected byhigh-performance liquid chromatography,we found that the content of GA3 and 6-BA in ZYX seedlings was higher than that of 02428,which reached a very significant level,suggesting that nal11 may have different regulation mechanism compared with severalreported narrow leaf gene which were associated with IAA,such as nal1,nal2,nal3,nal7and tdd1.2.Genetic analysis and preliminary mapping of nal11Through the CG1/ZYX F₂population,we found that some plants appeared showing narrow leaf,small panicle,thin and slender stems,low seed setting rate phenotype in the F₂population.After statistics,result showed that the number of plantswith wide leaves,stout stem,large panicle,high seed setting rate phenotype was 3 times of the number of plants showed narrow leaf,small panicle,thin and slender stems,low seed setting rate phenotype.This result showed that the narrow leaf,small panicle,thin and slender stems,low seed setting rate phenotypewas controlled by a single recessive gene,and this gene was named nal11.Through molecular marker linkage analysis,we located nal11 in the region of1487kb between RM21103and RM21196,the genetic distances were5.76cM and 46.62cM,respetively.3.Fine mapping of nal11By developing new makers and change the mapping population with a larger genetic distance parentcross 02428/ZYX,the narrow leaf gene nal11 was finally mapped between N10 and InD5016 with genetic distances of 0.75cM and 0.19cM respectively,the marker interval was a 58.3kb DNA region.4.Candidate gene prediction and analysisGene prediction of the 58.3kb target region was carried out by the rice genome annotation database,and the results showed that there were 9 candidate genes.We performed sequence analysis of these 9 candidate genes,found that only 2 genes exist differences:in the promoter region-162 ofOs07g09410there was an SNP;a single base mutation（G→T）occured in Os07g09450 encoding region.By sequencing other 11 rice varieties,we found that the single base mutation（G→T）occured inOs07g09450was specific.Thus,wetemporarilydesignatedthe putative heat shock protein DNAJ gene Os07g09450 as a candidate gene ofnal11.By analyzing the structure of Os07g09450,we found that the mutationin Os07g09450of ZYX was justoccurred on the last base in the second exon.5.Cloning and expression analysis of candidate genesBy5’and 3’RACE,we obtained the full-length cDNA of 02428 and ZYX gene Os07g09450.Sequence comparison showed that there are two kinds of transcripts in 02428,compared with 02428-cDNA1,02428-cDNA2 has a 35bp insert,however,the amino acid encoding by 02428-cDNA1 was 36 amino acids more than which 02428-cDNA2 encoding;due to the G→T mutation,an alternative splicing occured in ZYX’s mRNA,sequence originally belonging to the intron become part of the ZYX-cDNA,and then a stop codon behind the SNP resulting in premature termination of translation.Both 02428-cDNA1 and02428-cDNA2 encoding proteinscontain a DNAJ domain,SNP in ZYX’s mRNA leads to alternative splicing and early termination of the encoding protein without the DNAJ domain.6.Evolutionary tree analysis of NAL11Full length of NAL11’s mRNA is 861bp,encoding a 113 amino acidheat shock protein which composited a DNAJ domain.NAL11 is a highly conserved gene in the rice genome,there were highly homologous genes in maize,sorghum,soybean and Arabidopsis,all these highly homologous genes contain a DNAJ domain.Phylogenetic tree analysis showed that the rice NAL11 gene was most closely related with Brachypodium,wheat and barley,secondary closely related with maize,sorghum and millet,then followed by spruce,oil palm,cocoa,eelgrass,Physcomitrella patens and so on.7.Preliminary functional analysis of NAL11According to the results of pOX-ORF2overexpression vectortransformation experiments,we considered that NAL11-ORF2 could not restore the phenotype of02428-NIL.It is speculated that both structure and function of the 77 amino acids protein encoded by NAL11-OFR2 had been changed compared with 113 amino acids protein encoded by NAL11-ORF1.Histochemical staining of GUS transgenic plants showed that:NAL11expressed in rice root,leaf sheath,leaves,stem and panicle;fluorescence observation of GFP fusion expression vector transformated rice protoplastshow that:proteinencoded by NAL11/OsDNAJC1was localized in chloroplast.8.Expression profile analysisThe results obtained by RACE were verified by RT-PCR,real-time PCR and transcription sequencing.Through transcription sequencing we found 1209 differential genes,top 5 enrichment pathway mainly focus on RNA transport,mRNA surveillance pathway,biosynthesis of secondary metabolites,plant-pathogen interaction,andplant hormone signal transduction.These 5 enrichment pathwaywere consistent withheat shock protein function,molecular chaperone functionof NAL11 and cis-acting elements’function in NAL11’s promoter region.