Functional Analysis Of DCL,Which Located In The Centromeric Region Of Chromosome 8 In Rice

The centromere of eukaryotic chromosomes is essential for the faithful segregation and inheritance of genetic information.It is directly connected to the kinetochore microtubules during cell division,and is responsible for accurate sister chromatid cohesion and chromosome segregation in eukaryotes.For a long period,the centromeric region was considered to be highly heterochromatized and contained highly repetitive sequences,which were part of transcriptional silencing region in the chromosome.Meanwhile,centromeric and pericentric regions lack meiotic recombination.Therefore,it was generally considered that no functional gene was located in the centromeric region.However,active genes,which exist in the centromeric region,have been reported in recent years.Rice(Qryza sativa L.)is one of the most important food crops in the world,which has been widely used as a model organism for studying centromere.In addition,with the completion of the rice genome sequencing,the composition of active genes in different centromeric regions of chromosome was predicted.Among them,rice Centromere 8(Cen8)is the most ideal model system to reveal features of the centromeric region without the extensive satellite repeats,therein 16 active genes were predicted in Cen8 region.However,the function of active genes is few reported.In plant,the change of leaf color is resulted from both genetic and environmental factors in which genetic factors are usually related to the biosynthesis and degradation of chlorophyll in the formation and development of chloroplast and so on.In rice,leaf color mainly includes white,yellow,light green,yellow green,striped and so on.Numerous genes which are related to leaf color,distributed on each chromosome of rice.However,leaf color genes located on centromeric regions have few reported in rice.In this study,based on the material without centromeric repeats,derived from rice cultivar Zhongxian 3037,the material cannot grow normally in the field,and it is determined that there are signifant functional genes in the centromeric region.Development the research on the material,the 6 lost and differentially expressed genes in the variation centromeric region were analyzed and we focused on the analyzed the function of DCL(defective chloroplasts and leaves)in rice,which may a leaf color related gene,the results are as follows:1.FISH analysis showed that the deletion of the centromere sequence occurred on chromosome 8 without the CentO sequence of rice.2.Using PCR and RNA-seq analysis,the lost region in centromere of chromosome 8 was further identified between LOC_Os08g21541 and LOC_Os08g21710,its size is about 171kb,with 6 active genes,therein LOC_Os08g21700 is the candidate gene for DCL,which may a leaf color related gene.3.RNAi vector for the 6 genes were constructed,and the expression of DCL gene in the plants of RNAi was analyzed.The results showed that the expression of DCL gene was decreased significantly in RNAi plants.4.Phenotypic observations showed that the leaves of DCL-RNAi got etiolated and had significant changes on plant height,the number of tillers,seed setting rate and 1000-grain weight.5.The chlorophyll content was measured and the chloroplast structure was observed through electron microscopy.The results showed that the total chlorophyll content and chlorophyll components of DCL-RNAi was significantly decreased.It determination that the chloroplast formation and development were related with DCL gene.6.Genes related to the formation and development of chloroplast and photosynthesis in rice were selected to perform expression analysis in RNAi materials.Compared with wild type,only the expression of LOC_Os04g58200 encoding the NADPH chlorophyll oxidase was significantly decreased in the DCL-RNAi material,and the expressions of other genes in the wild type and DCL-RNAi materials have no significant difference.7.Expression analysis and GUS staining results showed that DCL gene was expressed in roots,stems,leaves and spikes of rice;the transient expression of GFP fusion protein in rice protoplasts showed that DCL protein was a nuclear protein.8.Over-expression and CRISPR/Cas9 vectors of DCL gene were constructed,and transgenic plants have been generated.The over-expression of DCL was also analyzed.The expression of DCL gene in hygromycin positive plants was significantly increased.