Analysis Of Agronomic Characters,Whole Transcriptome And Soil Microbiome Of Csu-miR260 Transgenic Rice

RNAi technology is widely used to inhibit or block gene expression due to its strong specificity and environment-friendly advantages.It also has good application prospects in the industrialization of transgenic plants.At present,there are no safety evaluation guidelines specifically for RNAi transgenic plants in China.In order to further refine the safety evaluation indicators of RNAi transgenic plants,this study carried out bioinformatics,agronomic characters,expression analysis of whole transcriptome and soil microbiology for RNAi transgenic plants represented by Csu-miR260 transgenic rice.The differences between the RNAi transgenic plant and the wild type were comprehensively compared,and the following results were obtained:(1)Bioinformatics analysis predicts that the exogenous transfer sequence of Csu-miR260 transgenic rice does not had complementary sequences to the rice genome,nor does it generate unexpected miRNAs,but there were potentially bound circ RNA.The transgenic insect-resistant rice had complementary sequences not only with the dib gene from target pest Chilosuppressalis,but also with Bombyx mori and other insects of the same genus as the stem borer.(2)Through the determination of agronomic characters,it was clear that transgenic rice Csu-miR260-16-4 and Csu-miR260-18-2 were significantly different from wild-type ZH11 in main agronomic characters,which were mainly reflected in the germination and maturity periods.During the germination period,the germination rate of ZH11 was 95.33%,that of Csu-miR260-16-4 was 62.67%,and that of Csu-miR260-18-2 was 40.67%.The germination rate of the two transgenic lines was significantly lower than that of ZH11.At the mature stage,the plant height of ZH11 was 106.54 cm,and the one of Csu-miR260-18-2 was shorter than that of ZH11,which was 96.14 cm.The number of grains per panicle of ZH11 at the mature stage was 89.41,and that of Csu-miR260-18-2 was 69.77,which was significantlyless than ZH11.The thousand-grain weights of the three varieties were different,Csu-miR260-16-4was 25.56 g,Csu-miR260-18-2 was 26.38 g,and ZH11 was 23.93 g.(3)Through the whole transcriptome sequencing and analyzing of two Csu-miR260 transgenic rice lines and three conventional rice ZH11,Nippon and Kita,it was determined that the gene expression differences of Csu-miR260 transgenic rice,which was obtained by Agrobacterium infection and callus seedling formation,were different from those of wild type ZH11 in mRNA,lnc RNA,circ RNA and miRNA,but the difference was much smaller than that among wild type rice varieties.Compared with ZH11,the number of differential mRNA of Csu-miR260-16-4 and Csu-miR260-18-2 were 723 and 1181,respectively,while the number of differential mRNA of Nippon and Kita were 1622 and 1295,respectively.The number of differential lnc RNA of Csu-miR260-16-4 and Csu-miR260-18-2 were 96 and 212,respectively,while the number of differential lnc RNA of Nippon and Kita were 261 and 206,respectively.The number of differential circ RNA of Csu-miR260-16-4 and Csu-miR260-18-2 were 71 and 67,respectively,while the number of differential circ RNA of Nippon and Kita were 89 and 69,respectively.The number of differential miRNA of Csu-miR260-16-4 and Csu-miR260-18-2 were 57 and 35,respectively,while the number of differential miRNA of Nippon and Kita were 123 and 140,respectively.It show that the effect of exogenous gene transfer on the expression of endogenous genes is less than the difference in expression of endogenous genes caused by different varieties.Aiming at the unexpected phenotype with low germination rate,the KEGG/GO enrichment analysis of differential RNA was carried out,and it was found that there were significantly different genes related to seed germination in hormone signal pathway,transcription factor and energy substance metabolism pathway.(4)Through the analysis of soil microbiome in the rhizosphere of wild-type ZH11 and two Csu-miR260 transgenic rice lines,it was determined that Csu-miR260 transgenic rice had effects on the community structure and composition of soil microorganisms,but the effects of the two transgenic lines were not consistent.In terms of fungal communities,the relative abundance of Ascomycota and Lipomyces of Csu-miR260-18-2 was different from that of ZH11,while that of Sordariales of Csu-miR260-16-4 was different from that of ZH11.Based on the above research results,the agronomic characters,gene expression characteristics at the whole transcriptome level and their effects on soil microorganisms of transgenic rice Csu-miR260-16-4 and Csu-miR260-18-2,had all been identified in this paper.The differences between Csu-miR260 transgenic insect-resistant rice and wild-type ZH11 were systematically analyzed from the aspects of bioinformation,agronomic characters,whole transcriptome gene expression and soil microbiome,so as to provide scientific basis and technical support for the refinement of safety evaluation guidelines for RNAi transgenic plants represented by Csu-miR260 transgenic insect-resistant rice.