| Csu-mi R260 transgenic rice has a good insect resistance effect and potential for industrial development,but the molecular characteristics of Csu-mi R260-16 and Csu-mi R260-18 strains are not clear,and they have phenotype with decreased germination rate.In this study,two independent Csu-mi R260-16 and Csu-mi R260-18 strains of Csu-mi R260 transgenic rice were used as research materials to analyze the flanking sequence characteristics of the foreign gene insertion site,genomic variation characteristics,the expression characteristics of mi RNA and precursors and the germination characteristics of seeds under different hormone treatments.It provides data support for the safety evaluation of the genetically modified product,and also provides a theoretical basis and reference for the safety evaluation of insect-resistant RNAi genetically modified crops.The main research results obtained are as follows:1.The T-DNA insertion sites of the two strains Csu-mi R260-16 and Csu-mi R260-18 were simultaneously identified using TAIL-PCR technology and genome resequencing.It was found that the left and right sides of the insertion site of Csu-mi R260-16 are 11863348 and 11863480 on chromosome 1,which replace the genome of 133 bp.The transferred T-DNA sequence has a 27 bp deletion on the left border and a 54 bp deletion on the right border.The left and right sides of the insertion site of Csu-mi R260-18 are 31311317 and31311395 on chromosome 4 respectively,which replace the genome of 78 bp.The transferred T-DNA vector sequence has a 170 bp deletion on the left border and a 24 bp deletion on the right border.2.Through genome resequencing to analyze the genomic variation characteristics and taking the Nipponbare genome as a reference,found that the numbers of SNPs in Zhonghua11,Csu-mi R260-16 and 18 were 194,172,193,030 and 193,289 respectively;In Dels were41218,40610 and 40551,respectively;SVs were 6,401,6287 and 6,241 respectively;CNVs were 4868,4794 and 3852 respectively.Secondly,it was found that the SNPs and In Dels of Zhonghua 11,Csu-mi R260-16 and 18 were all distributed at a higher density in the middle of chromosome 6 and the end of chromosome 11.After filtering the different SNPs and In Dels using the Zhonghua 11 and 3000 rice resequencing databases,it was found that the unique SNPs and In Dels in Csu-mi R260-16 and 18 were 17688 and 10763,respectively,and the shared SNPs and In Dels were 9742 and 6036,respectively.The filtered SNPs and In Dels mutations in Csu-mi R260-16 and 18 all have a higher density distribution at the end of chromosome 11.3.TaqMan probe real-time fluorescent quantitative PCR(TaqMan RT-q PCR)and stem-loop real-time fluorescent quantitative PCR(Stem-Loop RT-q PCR)were used to quantitatively detect the expression of the Csu-mi R260 insert sequence and the sheared ami R260 s in Csu-mi R260-16 and Csu-mi R260-18 transgenic rice,respectively.It was found that the insertion sequence of Csu-mi R260 and the 20 nt and 21 nt mature ami R260 s formed by shearing were expressed in the roots,stems and leaves of the transgenic insect-resistant rice seedlings and had no tissue expression specificity.4.Through seed germination experiments,it was found that the germination rates of Zhonghua 11,Csu-mi R260-16 and Csu-mi R260-18 were 95%,60% and 40%,respectively.The germination treatment of Zhonghua 11,Csu-mi R260-16 and 18 with 1 μM ABA showed that the germination rate of Zhonghua 11,Csu-mi R260-16 and 18 all decreased,and when10 μM ABA was used,the germination rate was lower.When 1 μM and 5 μM GA were used It was found that 1 μM and 5 μM GA had a significant promoting effect on Csu-mi R260-16,but not Csu-mi R260-18.Besides when ABA inhibitors were used,it was found that 1 μM and 5 μM ABA inhibitors both promoted the germination of Csu-mi R260-16 and 18.5.According to the characteristics of insertion site flanking genes of Csumi R260-16 and Csumi R260-18 transgenic rice and with differential expression,two CRISPR/Cas9 knockout transgenic rice materials with abnormal expression of the two genes LEA9 and STPK in the flanking sequence were constructed,and obtained transgenic rice lines with editing effect. |
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