Study On The Function Of LncRNA565 In Regulation Of Rice Seed Vigor

Rice(Oryza sativa L.)is one of the world’s major food crops,more than half of the world population live on rice.Rice seed aging can lead to abnormal seed germination and growth,which seriously affects grain yield.Long non-coding RNA(lncRNA)can regulate plant growth and development in various ways,however the current research and understanding of its function in seed vigor are extremely limited.Some differentially expressed lncRNA,including lncRNA565,were screened from rice seed aging transcriptome in our laboratory,and it was preliminarily speculated that lncRNA565 may play an important role in the regulation of rice seed vigor.Thus,in this thesis,taking two rice varieties Nipponbare(O.sativa L.spp.japonica)and Dongjin(O.sativa L.spp.japonica)as materials,the expression pattern of lncRNA565 and its variation characteristics in the aging process of rice seeds were investigated.Additionally,the CRISPR/Cas9 mutant and T-DNA insertion mutant of lncRNA565 were used to further study their possible functions in seed vigor regulation.The main research results were as follows:1.LncRNA565 was found to be located on chromosome 1 of Nipponbare,and it was confirmed that lncRNA565 was a long non-coding RNA based on the analysis of Fickett score,isoelectric point,and peptide length.2.LncRNA565 had obvious tissue-specific expression in Nipponbare,with the highest expression level in 25-days-after-pollination embryos,followed by leaves,and then in 5-days-after-pollination seeds,roots,seedlings and stems,and in flowering inflorescences.3.Thirty-three lncRNA565 knockout mutants of T0 generation were obtained by CRISPR/Cas9 technology and genetic transformation,and 24 positive seedlings were screened from them,including 3 homozygous mutants,8 biallelic mutants,3 heterozygous mutants and 10 wild-type strains.In addition,one T-DNA insertion mutant strain was ordered in the rice mutant database,and the homozygous mutant strains were screened out.4.The germination rate of the knockout mutants lncrna565-2 and lncrna565-32 seeds after artificial aging for 9 d were significantly higher than those of the wild-type,and the expression of lncRNA565 in both strains was significantly down-regulated by 91.4% and76.3%,respectively.5.The expression of lncRNA565 in T-DNA insertion mutant lncrna565-T was significantly up-regulated.Additionally,the expression of lncRNA565 in knockout mutants lncrna565-2 and lncrna565-32 were significantly down-regulated,and the seed length became shorter.6.ZOS1-22 was a co-localization gene of lncRNA565,and the expression patterns of ZOS1-22 in various tissues of Nipponbare were similar to those of lncRNA565,with the highest expression in embryos,followed by leaves.Moreover,ZOS1-22 could responds to artificial seed aging treatment.In addition,the expression patterns in lncRNA565 mutants were also similar,which were down-regulated in knockout mutants lncrna565-2 and lncrna565-32,and were up-regulated in T-DNA insertion mutant lncrna 565-T.