Preliminary Study On The LncRNA-mediated Mechanisms During Seed Maturation In Arabidopsis Thaliana

Seed maturation is a special and complex development process,which is not only a critical period to determine the quality and yield of seeds,but also an important link to affect the quality of seed storage and preservation.However,the understanding of the regulation mechanism of seed maturation is still relatively weak.LncRNAs?Long noncoding RNAs?,which vary in length from 200 nt to dozens of kilobases,are a class of noncoding RNAs.Recent studies have shown that they are abundant and widely distributed in eukaryotes,and can regulate gene expression or directly affect protein function at epigenetic,transcriptional and post-transcriptional levels.At present,people have a certain understanding of the function and mechanism of plant lncRNAs,which play an important role in many biological processes.However,there is no report on the function and mechanism of lncRNAs in seed maturation.In this study,we identified the lncRNAs mediating the regulation of seed maturation from the perspective of seed storage proteins by using the identification system of seed maturation regulators established in the laboratory and preliminarily elucidated their mechanisms.The main findings are as follows:1.The seeds of Arabidopsis thaliana at four developmental stages?4,7,12,17DAP?were collected.A total of 64 candidate genes of lncRNAs responding to seed maturation were obtained by high-throughput detection and bioinformatics analysis.Among them,24 were up-regulated genes and 40 were down-regulated genes.24lncRNAs located in the gene transcription module region,35 were intergenic lncRNAs and 5 were antisense lncRNAs.2.The selection of the 9 lncRNA genes were cloned and constructed into expression vector,and then six up-regulated lncRNAs were transformed into gus23?seed maturation reporter gene system?and three down-regulated lncRNAs were transformed into wild-type Arabidopsis thaliana by flower dipping infection,genetically modified homozygotes were obtained for subsequent research.3.GUS staining was used to analyze whether six genes transferred into gus23were involved in the regulation of seed maturation.The results showed that lncRNA-Seed9 and lncRNA-Seed53 could induce GUS phenotype in the true leaves of gus23 seedlings,suggesting that SSPs of seed storage protein genes were heterotopically expressed in two seedlings with over-expression of lncRNA.Subsequently,qPCR results showed that the expression levels of seed maturation regulator LEC2 and storage protein 2S2 genes in lncRNA-Seed53 over-expressed seedlings were significantly higher than those in wild-type seedlings.The expression of major regulators of seed maturation in seeds of over-expressed plants was further detected.The results showed that LEC2 gene was up-regulated slightly in siliques of over-expressed plants with lncRNA-Seed53 for 7 DAP,and the expression of LEC1gene in 12 DAP siliques was significantly increased.These results suggest that lncRNA-Seed53 is a regulator of seed maturation.4.The phenotypes of seeds and other aspects of transgenic plants were analyzed.The results showed that over-expression of lncRNA-Seed53 resulted in short and full siliques,slow silique growth and large seeds.In addition,there were short petioles,round and small lotus leaves,early flowers,compact buds,shorter petals and other phenotypes in seedling stage.At the same time,we also observed that the seed germination of over-expressed lncRNA-Seed26 was inhibited,and the plant had the phenotype of late germination and small seedling plant.5.In order to elucidate the regulatory mechanism of lncRNA-Seed26 and lncRNA-Seed53,preparations and preliminary exploration were made.1)The expression of adjacent genes of two lncRNAs was analyzed by qPCR.The results showed that KIP1 gene expression was suppressed by lncRNA-Seed53,TPPI gene expression was increased in over-expressed seedlings of lncRNA-Seed26,and LRR gene expression was down-regulated.2)In order to further study the functions of these lncRNAs,we designed two gRNA targets for lncRNA-Seed26 and lncRNA-Seed53 respectively,constructed two double gRNA target saCas9/gRNA vectors and transformed them into Arabidopsis thaliana.Seven lncRNA-Seed26-CRISPR mutants were screened and identified,the editing efficiency of saCas9/gRNA vector was 2.083%.and that of three lncRNA-Seed53-CRISPR mutants was 0.704%.3)We constructed 35S:S1 streptavidin-tagged lncRNA-Seed26 vector by bioinformatics analysis and overlapping extension PCR,and screened and identified T₁ generation S1-lncRNA-Seed26 transgenic plants,which laid the foundation for identifying the target gene of the lncRNA.These results have broadened people's understanding of the regulation network of seed maturation and the biological functions of plant lncRNAs,and laid a foundation for further research on the regulation mechanism of these lncRNAs.