Phenotypic Analysis And Mutant Gene Identification Of Rice Yellowing Mutant Crt1

Rice is not only the main food crop for 1/2 people in the world,but also a model plant for Monocotyledon research.Leaf color mutation is a common mutation type in rice,which can be used to solve hybrid pollution in cross breeding,save cost,provide material source for rice breeding with high light efficiency,and improve rice yield and regional adaptability.it is of great significance in basic research and production application.Etiolated mutant crt1 is a mutant selected by our group in the process of rice callus transformation,which results in leaf color change caused by T-DNA insertion.In this paper,the phenotype of the mutant was analyzed and the mutant gene was identified.The main results are as follows:(1)By observing the field phenotype of mutant crtl under natural light in Changsha,it was found that the mutant leaves were yellow during the whole growth period from seedling growth to heading.The detection results of leaf pigment content at tillering stage showed that compared with the wild type,the content of chlorophyll an and carotenoid in the mutant crt1 decreased by 31%and 25%respectively,the total chlorophyll content decreased significantly,and the SPAD value decreased significantly,but the ratio of chlorophyll to carotenoid did not change significantly.The results of leaf net photosynthetic rate and stomatal conductance showed that the net photosynthetic rate and stomatal conductance of crtl were significantly lower than those of wild type,indicating that etiolation led to the weakening of photosynthesis.(2)The results of agronomic characters of etiolated mutant crt1 showed that compared with the wild type,the plant height,ear length,flag leaf length and width of the mutant increased by 33.39%,28.06%,51.91%and 11.76%,respectively,and the heading period was prolonged for 24 days.However,the effective tiller number,seed setting rate and 1000-grain weight of the mutant decreased by 42.42%,29.43%and 10.75%,respectively.(3)The scanning results of transmission electron microscope showed that compared with the wild chloroplast morphology,the number of chloroplast grana of mutant crt1 was significantly reduced,no complete photosynthetic membrane system was formed,and the structure changed obviously.(4)The content of intermediate metabolites in chlorophyll synthesis showed that the content of ALA in mutant crt1 increased significantly,while the content of PBG and ProtoIX decreased significantly,but the content of UrogenⅢ,CoprogenⅢ,Mg-ProtoIX and Pchlide did not change significantly.qRT-PCR analysis of chlorophyll synthesis related genes at transcriptional level showed that the expression of HEMA1,POPB and CHLH decreased significantly,while the expression of HEMC,HEME2,HEMF1 and CHLG increased significantly,while the expression of HEMB1,HEMD.HEME1,POP A,CHLD,CHLI,CHLM and GSA1 did not change significantly.The results showed that the chlorophyll synthesis in the mutant crt1 was seriously affected and was related to the expression of genes related to its synthesis.(5)The T-DNA flanking sequence was amplified by Genome walking,and it was found that the flanking sequence was highly homologous to the OsDHHC24 gene sequence.The results of bioinformatics analysis showed that OsDHHC24 encodes a zinc finger protein with DHHC conserved domain and has four transmembrane domains.OsDHHC24 was more transcribed and expressed in young roots,inflorescences and panicle tips of rice,but less in leaves.OsDHHC24 promoter analysis showed that the pre-1500bp promoter sequence contained several cis elements,such as 3AE1bindingsite,ACE,AE-box,CGTCA-motif,TGACG-motif and so on,suggesting that OsDHHC24 may be involved in photosensitive and multi-hormone regulation in rice.(6)The Ubi::OsDHHC24-GFP vector was constructed and the positive Ubi::OsDHHC24-GFP/crt1 complementary plants were obtained by transforming the etiolated mutant crtl callus.At the same time,the knockout vector of CRISPR/Cas9 of OsDHHC24 was constructed to observe whether the phenotype of complementary plants was restored or not,and to knock out the phenotype of mutants,so as to further determine whether OsDHHC24 is the key gene causing etiolation of mutant crt1.In short,a new rice yellowing mutant was discovered and identified,and its mutant gene was preliminarily identified,which provided a new research material and preliminary basis for further analysis of the developmental mechanism of chloroplasts.