Origination And CRISPR/Cas9 Mutants Generation Of A New Gene In Arabidopsis Thaliana

The origin of new genes is a fundamental phenomenon in the evolution of species in nature.Gene duplication is the main way of the origin of new genes through DNA-based mechanism,and retroposition is an RNA-based duplication.Here,We investigate the evolution and biological function of Arabidopsis thaliana gene AT3G11810 by synteny analysis,population genetics and phylogenetic analysis.The main results are as follows:1.Molecular mechanisms of the origin of novel genesThrough orthologs and paralogs gene alignment and synteny analysis of A.thaliana gene AT3G11810 and AT2G03330,Arabidopsis lyrata gene AL5G13040,Capsella rubella gene Carub.0005s0231,Brassica rapa gene Brara.I02088 and Eutrema salsugineum gene Thhalv10005445 m,I found that AT3G11810 is a new gene produced by retrotransposition.After the divergence between Arabidopsis and Capsella(about 10 MYA),mRNA produced by transcription of the old gene AT2G03330 was synthesized into cDNA by reverse transcription and inserted into the genome to produce the new gene AT3G11810.2.Population genetic analysis of novel genesDNA Polymorphism,nonsynonymous and synonymous analysis and neutrality tests of AT3G11810 and AT2G03330 from 855 accessions of A.thalianaworldwide were analyzed.The results of DNA polymorphism analysis showed that compared with AT2G03330,AT3G11810 contains fewer polymorphic sites and has lower DNA polymorphism,which suggests that AT3G11810 may be affected by natural selection.The ratio analysis of nonsynonymous rate to synonymous rate(Ka/Ks,ω value)showed that compared with the old gene,the ω value of the new gene AT3G11810 was 1.6085,indicating that the new gene AT3G11810 may be subjected to positive selection(ω>1).The results of the neutrality test showed that the origin of the new gene AT3G11810 was significantly different from the theoretical model of neutral evolution,and the origin and spread of AT3G11810 in the natural population were probably driven by positive selection.3.Function exploration of new and ancestral genesRT-PCR was performed on new gene and old gene using different tissues to generate gene expression profiles.The expression patters of AT3G11810 and AT2G03330 were commonly similar by highly expressing in the leaves,stems,roots,flowers and young fruits.In order to further study the biological function of the new gene and its ancestral genes,we screened homozygous T-DNA insertion mutants.The growth rate and flowering time of the wild-type and AT3G11810 T-DNA homozygous mutants were compared.It was found that there was no difference in the flowering time of the two strains under long daylight conditions(light for at least 12 hours).The flowering time of the wild type and the mutant was significantly different when cultured under the short-day condition of 8 hours of light and 16 hours of darkness.The average flowering time of the wild type was about 56 days,while that of the mutant was significantly extended to 69 days,indicating that the AT3G11810 gene had a significant phenotypic effect on flowering time of A.thaliana.4.Construction of CRISPR/Cas9 mutants of new and old genesNew and old gene mutants were constructed using CRISPR/Cas9 gene editing technology.After Agrobacteria-mediated transformation of wild type Arabidopsis thaliana,the strong promoter PcUBI in the expression vector drives Cas9 expression in A.thaliana and establishes the transformation system.Hygromycin was used to screen positive seedlings from T0 generation A.thaliana seeds.Finally,three CRISPR mutant lines of old and new genes were obtained,respectively.The results of sequencing and sequence analysis showed that base deletion,substitution or insertion occurred in the coding regions of the new and old genes.This study provides a new case of the molecular mechanism and evolutionary history of young plant genes in the early stage of origin,and enriches the model of the retention and further differentiation of duplicate genes driven by natural selection.