杨树花发育基因PagAG2基因编辑突变体创制及其基因组变异分析

[Objective]Trees play an important role in improving the level of urban greening,but po llen scattered by male plants causes certain plant-derived pollution,which is one of the important allerge ns for human body.It is urgent to cultivate environmentally friendly poplar varieties with little or no pol len to improve the pollen environment in northern cities.[Method]The key gene PagAG2 in male flowe r development was accurately edited using CRISPR/Cas9 gene editing technology,and the expression lev els of the target gene and its upstream and downstream related genes were analyzed by real-time quantit ative PCR.Sanger sequencing and WGS techniques were used to evaluate the off-target situation of gene editing and the effect of the gene editing event on the whole genome variation.Transcriptome and prot eome high-throughput sequencing techniques to evaluate the role of PagAG2 mutation in edited plants in regulating key processes of vegetative growth.[Result]In this study,CRISPR/Cas9 gene editing technolo gy was used to obtain poplar PagAG2 gene editing plants,which caused 6 bp base deletion and 2 bp b ase substitution near the target site.The expression of PagAG2 gene in the root,stem and leaf of the e dited plants was lower than that of the wild-type,and the growth rate was higher than that of the wild-t ype.Meanwhile,PagAG2 mutation can regulate the expression of its downstream genes(STK1,STK2,SP L16,SPL25),but has little effect on the upstream gene AP2,which may have an antagonistic effect on t he AG gene.The analysis results of Sanger sequencing and WGS on gene editing off-target showed that the sgRNA designed in this study had high specificity and no off-target event was found.The genome v ariation of wild-type plants was similar to that of gene-edited plants,and most of the mutations were sh ared by the wild-type plant and the gene-edited plant(SNPs was 55.9%,indels was 36.1%),which was mainly due to somaclonal variation in tissue culture.Cas9-induced mutations showed high specificity in Populus alba×P.glandulosa'84K'.The differences in transcription and protein levels between wild-typ e plants and gene-edited plants were mainly due to spontaneous mutations and the mutations induced by Cas9 endonuclease.The results of the multi-omics analysis showed that the genes corresponding to the v ariation on the genome exon,the differential genes of the transcriptome and the differential proteins of t he proteome were mainly enriched in biological pathways such as flavonoid biosynthesis,circadian rhyth m-plant,and galactose metabolism,which may also be the reason for the phenotypic differences between wild-type plants and edited plants.[Conclusion]In this study,CRISPR/Cas9 gene editing technology was used to create a homozygous mutant of the key gene of male flower development,and it showed no a dverse effects in the vegetative growth stage,and the gene editing event did not have a significant effect on the genome variation of the edited plants,which laid an important foundation for the use of gene e diting technology to develop environmentally friendly poplar varieties with no pollen or little pollen.