Acquisition Of HyPRPs Gene Edited New Poplar Germplasm And Analysis Of Drought And Salt Tolerance Mechanism

Drought and salinity seriously restrict the growth of plants,with the change of climate,the situation of land salinization and drought is severe,there is an urgent need to cultivate new resistant tree species.In this study,using CRISPR/Cas9 gene editing technology,Populus alba×P.glandulosa（84K poplar）and Populus×euramaricana‘Bofeng 3 hao’were used as research materials.Through the establishment of efficient genetic transformation system and gene editing vector construction and optimization,the multi-site genome of poplar was carried out.Through comprehensive evaluation of drought and salt stress resistance and molecular mechanism analysis of transcriptome-level stress resistance,6 gene-edited poplar lines with significantly improved stress resistance traits were successfully obtained.The specific research results are as follows:（1）5 negative genes related to drought and salt tolerance in poplar with potential application value were successfully cloned.Based on the amino acid sequence of tomato drought and salt negative regulator Sl Hy PRP1,two alleles Pag Hy PRP1A and Pag Hy PRP1B（Pag Hy PRP1）and 16 homologous genes were screened from the genomes of 84K and Populus deltoides respectively.q RT-PCR analysis showed that Pag Hy PRP1 was highly expressed in the roots of 84K poplar,and Pd Hy PRPs were mostly highly expressed in the roots and stems of P.euramaricana‘Bofeng 3 hao’.After drought and salt stress,Pag Hy PRP1（Pag Hy PRP1A and Pag Hy PRP1B）,Pd Hy PRP1,Pd Hy PRP2 and Pd Hy PRP8 were down-regulated in roots and stems.Therefore,these five genes were successfully cloned as candidate genes for negative regulation of drought and salt stress.（2）Based on transgenic and CRISPR/Cas9 technology,12 overexpressing lines（OE）of84K were successfully obtained,and 15 lines were positive for gene editing,among which 9lines of Pag Hy PRP1 were edited,including 2 homozygous mutants,2 biallelic mutants,and 5chimeric mutants,with editing efficiency of 60%.4 paghyprp1（prp-1,prp-2,prp-4,prp-6）edited lines with both Pag Hy PRP1A and Pag Hy PRP1B knocked out were screened for stress tolerance.The results showed that the plant height,ground diameter,stem weight,root weight and root shoot ratio of paghyprp1 edited lines were significantly higher than control（WT）.The activities of superoxide dismutase（SOD）and peroxidase（POD）and the content of proline were significantly higher than control.The accumulation of hydrogen peroxide（H₂O₂）,superoxide ions（O₂.^-）,malondialdehyde（MDA）and reactive oxygen species（ROS）was significantly lower than control.The edited lines also had higher Na⁺efflux（under salt stress）,Ca²⁺efflux（under drought stress）and H⁺efflux,but less K⁺efflux.The indexes of OE lines showed opposite results,and there was no significant difference between edited lines.The results indicated that paghyprp1 edited lines could improve the drought and salt tolerance of plants by increasing antioxidant enzyme activity,reducing ROS accumulation,reducing the degree of membrane peroxidation,maintaining root ion balance,and stabilizing root osmotic balance.（3）Transcriptome sequencing analysis showed that:compared with WT,340 significantly differentially expressed genes（DEGs）were screened in the stress response hormone,growth promoting hormone,ROS production and photosynthesis-related pathways in prp4 and OE1lines under salt stress,indicating that deletion and overexpression of Pag Hy PRP1 gene affect salt tolerance mainly by regulating key gene expression changes in the above pathways.Further,6 key up-regulated genes and 30 key down-regulated genes regulating ROS production were identified by WGCNA analysis.In addition,protein interaction network analysis identified 10 key genes involved in MAPK signaling pathway,which were also involved in multiple signaling pathways such as ABA,H₂O₂,flg22 and Ca²⁺,and were lowly expressed in prp4 line and highly expressed in OE1 line.The above key genes for salt stress response are important for prp4 line to improve salt tolerance.（4）The genetic transformation systems of P.euramaricana‘Bofeng 3 hao’leaf disk method and callus method were established,and the conversion efficiency was 11.47%and16.54%,respectively.5 overexpressing Pd Hy PRP2 lines,2 inhibiting Pd Hy PRP1 and Pd Hy PRP2 expression lines,and 2 mutant Pd Hy PRP1 and Pd Hy PRP2 gene editing lines were successfully obtained.Under salt stress,the contents of O₂.^-,H₂O₂and MDA in leaves and roots were the lowest,which were 0.60-0.85（leaves）and 0.66-0.86（roots）of wild type P.euramaricana‘Bofeng 3 hao’,indicating that the gene edited line could reduce oxidative damage in leaves and roots and improve salt tolerance.