Analysis Of Endophytic Microbial Variation In Transgenic Rice Seeds

With the increase in the types and area of planting of genetically modified crops,the issue of safety of genetically modified crops has attracted widespread attention.As one of the world's major food crops,the important part of evaluating the safety of genetically modified rice is to assess its unintended variation.Endophytic microorganisms are including bacteria and fungi groups that implanted in healthy palnt,the variation in structure and distribution of endophytic flora in rice seeds can have an important effect on their food and ecological safety.The differences of the endophytic bacteria between japonica rice Nipponbare,D349 and indica 28 strains of GM and non-GM rice were compared and analyzed,using 2mG2-epsps transgenic rice as materials.And the differences in culturable endophytic fungi of Nipponbare and D349 lines of transgenic and non-transgenic rice seeds and their dynamic changes with storage time were monitored under two storage conditions.A pair of primers suitable for analysis of endophytic bacteria in rice seeds were also screened out and used to analyze the structure of the endophytic bacteria floria of Nipponbare rice seeds from the meta-genomic level.The main results are as follows:(1)There is a great deal of instability in the detection of endophytic bacteria in rice seeds.After repeated detection of 15 times,the species of endophytic bacteria can be stable.Seven strains of endophytic bacteria were isolated from all rice seed samples,The representative strains were identified as Pantoea,Pseudomonas,Leucobacter,Enterobacter,Bacillus,Bactirum,Microbacterium,and the dominant genus of rice seeds was B1(Pantoea)and B5(Bacillus).Comparing the three rice lines,it was found that the colonization of endophytic bacteria in rice seeds of different lines was significantly affected by the 2mG2-epsps gene.The increase in the colonization number of endophytic bacteria in rice seeds of P1 and P3 lines was greatly influenced by the insertion of 2mG2-epsps gene,while the colonization number of endophytic bacteria in P2 rice seeds was greatly affected by tissue culture;gene insertion increased B2(Pseudomonas)colonization in rice seeds,and the colonization of B1(Pantoea)in P3 is significantly increased,whereas colonization of B1(Pantoea)in P2 line rice seeds is afected by tissue culture effect,making it set the amount of colonization increased significantly,gene insertion and tissue culture significantly inhibited colonization of B5(Bacillus)seeds in P3 lines.(2)Using the PTN system to trace the sources of endophytic bacterial colonization of the GM rice seeds using three lines of transgenic rice seeds,it was found that the cultivatable fungal variation of the GM rice seeds of the P1 line may be affected mainly by gene insertion and tissue culture in the transgenic process.However,the cultivatable fungal variation of P2 and P3 strains of GM rice seeds may be mainly affected by tissue culture during the transgene process and is less affected by gene insertion.(3)The traditional isolation and culture method was used to track and monitor the changes of the bacterial groups of endophytic fungi in Nipponbare(P1)and D349(P2)lines under both room temperature and 4? storage conditions.47 endophytic fungi were isolated from rice seeds and belonging to 22 genera and 47 species.The dominant groups were Dendryphiella,Curvularia and Fusarium.The relative isolation frequencies were 59.01%,15.51%and 8.16%,respectively.Comparing the two storage conditions,it was found that the isolated rate and species number of rice seeds under 4? storage conditions were higher than room temperature conditions,and the 4? storage conditions could better maintain the activity of the fungi in rice seeds.Comparing the two rice lines,it was found that the insertion of 2mG2-epsps gene increased the colonization and the species number of endophytic fungi in rice seeds,which increased the bacterial colonization rate;with the increase of seed storage time,both the carrier rate and the detected species of GM and non-GM rice seeds showed a downward trend.The similarity between the GM and non-GM of the P1 line gradually increased,and the similarity of the P2 line gradually decreased.The colonization of endophytic fungi in different rice lines was greatly influenced by the influence of 2mG2-epsps gene.(4)The primer sequences of 7 pairs of high-throughput sequencing bacterial PCR primers were verified by high-throughput sequencing through blast homology alignment and Primer-blast alignment with rice genome.It was found that primers pairs Pr7(fMl and rCl)were more suitable for high throughput PCR amplification as primers of endophytic bacteria of rice seed,which could reduce the interference of rice organelle genes.Illumina MiSeq high-throughput sequencing of primers Pr7 was used to analyze the endogenous bacterial community structure of Nipponbare seeds,and the endogenous bacterial groups of Nipponbare rice seeds were classified into 62 OTUs,which were divided into 6 phylums,10 classes,17 orders,23 families and 37 genus.The dominant bacteria were Pantoea,Candidatus Kuenenia and Lactococcus,accounting for 3.19%,0.09%and 0.06%,respectively.