Analysis Of Rice Mutant RMIM91 WGS And RNA-Seq Induced By Maize DNA

Exogenous DNA import is a new way to create new germplasm and cultivate new varieties,mutant materials were obtained by introducing foreign DNA into cells of the plant germplasm system,and new germplasms and new varieties with certain target traits were selected.The laboratory used a modified "pollen tube mediation method" to introduce maize DNA into rice "Nipponbare" and obtained a batch of mutant strains.After several generations of self-cross selection and breeding to the eighth generation,a stable genetic variation line that differed significantly from Nipponbare agronomic traits was selected and designated as RMIM91(hereinafter referred to as R91).The whole genome re-sequencing,transcriptome sequencing,protein structure prediction and hormonal signal transduction pathway analysis were performed on R91.The results showed that: 1.A large number of mutation sites were detected in the genome sequencing results.After comparison with the rice database and control genomic data,a total of 200,479 R91-specific mutation sites,including 103,314 SNPs and 97,165 In Dels,were screened,indicating that mutagenic effects of foreign DNA on rice genomic DNA were significant.2.There were 62,647 bases in R91-specific SNP mutations and 40,667 base inversions.The In Del mutations included 61,816 insertions and 35,348 deletions,indicating that R91 has a greater frequency of transversion than transversion,and more insertion mutations than deletion mutations.3.In R91,the frequency of mutations in genomic bases ranges from high to low :G(30.76%)>C(30.28%)>A(19.59%)>T(19.37%),indicating that G and C are susceptible to mutations in the R91 generation.4.In the SNP mutation site,there are 26,453 mutation sites for PuNPu in the adjacent side,26,314 mutation sites for PyNPy,and 50,545 mutation sites for PurNPy&PyNPu in the adjacentside,among which the base There were 8,610 mutation sites for CNG with the highest frequency of mutations,indicating that the bases are more susceptible to mutations when the adjacent bases are C and G.5.There were 43 SNP mutations at key positions in the gene(1 gene promoter deletion mutation,17 terminator mutations,and 4 splicing mutations),there are 5 mutations had detected in R91 the next generation.A total of 34 SNP mutations were detected in the known functional genes and upstream or downstream,respectively,located in 10 genes,and 4 of them affected protein codes,of which 2 mutations were re-detected in the R91 generation.This indicates that the mutation site detected by whole genome sequencing does exist in R91,and introduction of exogenous DNA can induce SNP mutation in the rice genome.6.There are 986 InDel mutations located in the key positions of genes,of which 613 are inserted and 374 are missing.The insertion and deletion bases are mainly distributed in the range of-2~2 bases,It indicates that In Del mutation is mainly inserted and missing below 3 base.7.InDel mutations were screened out of 25 In Del sites for detection.There were 14 mutations detected in the next generation of R91;there were 53 known function genes and upstream and downstream InDe mutations,affecting Of the18 genes,six mutations were detected and re-detected in the next generation of R91.This indicates that introduction of exogenous DNA induces InDel mutations in rice.8.Analysis of transcriptome data of flag leaf and stem sections at the flowering stage of the R91 generation showed that the expression levels of some mutant genes were significantly different from those of Nipponbare: Os06g0240400 and Os12g0116200 were silenced in R91,and Os05g0309000 was the gene that was activated and expressed.Os06g0246200;Os01g0686200 gene expression in stem nodes and flag leaves increased,Os09g0567000 gene expression in the stem node and flag leaf expression was down,Os05g0390500 and Os08g0193300 gene expression in the stem nodes only upregulated,Os05g0309000 only flag leaf expression was upregulated,Os10g0126500 gene only stem Festival downgrade.It indicates that the mutation induced bythe introduction of exogenous DNA can affect the expression level of rice genes during flowering stage.9.Protein structures prediction analysis results show that 27 heavy in the detection of mutations,there are 7 damages to the structure and function of the protein that mutations can caused by exogenous DNA into a larger influence on protein function.10.Function rich in cluster analysis showed that abscisic acid and ethylene signaling pathways in the part of the significant difference,the level of gene expression showed that exogenous DNA into induced mutations have an impact on the two hormone signaling pathways.The above results indicate that the introduction of exogenous DNA is an effective biological mutagenesis technique that can induce a large number of SNP mutations and indel mutations in rice genomes,as well as the insertion of a small number of foreign DNA sequences,change rice gene sequences,and encode genes.The protein structure and function as well as gene expression levels has a major impact.The easily mutated bases and mutation-prone sites found in the study provide a theoretical basis for studying the molecular mechanism of rice gene mutations;multiple mutation traits induced by mutagenesis,new gene discovery and functional gene research,and creation of new germplasm resources And the use of significance.