Creating Low-Gossypol Cotton New Germplasm Via CRISPR/Cas9 And Study On Transcription Factors Regulating Gossypol Biosynthesis

Not only cotton fiber showed wide application,but also other by-products showed highly utilization value,such as cottonseeds.Cottonseeds are rich in protein and oil,but the presence of gossypol poses a health concern for both domestic-animal and human uses.Researchers have been aiming for the cultivation of low-gossypol cotton,the biosynthetic pathway of gossypol has been elucidated in recent years,the（+）-δ-cadinene synthase encoded by the CADS gene is a rate-limiting enzyme in the gossypol biosynthetic pathway,knocking out the CADS gene via CRISPR/Cas9 is an effective method to prevent the synthesis of gossypol and improve the efficient utilization of cottonseed.Therefore,the creation of new cotton germplasm with low gossypol in seed by CRISPR/Cas9 has important production and application value.In addition,combined with RNA-seq data,we analyzed the regulation of MYC2-D09and WRKY1-D11 transcription factors in response to jasmonic acid on gossypol biosynthesis.The results were as following:1.A method for detecting CRISPR vectors in cotton hairy roots was established.The efficiency of Agrobacterium rhizogenes K599 with four OD600（0.4,0.6,0.8 and 1.0）in inducing hairy roots was compared,and the bacteria with OD600=0.8 showed the highest induction rate（30%）.Different types of cotton cultivars were induced hairy roots production by the method.After K599 bacteria harboring the CRISPR plasmid were injected into cotton top buds to produce hairy roots,mutations in the target genes（GhPDS and GhMYB25-like）were detected in the hairy root DNA.This method could be used to evaluate target sites and CRISPR/Cas9 vectors,and provides help for getting editing vectors with high efficiency.2.According to the expression pattern of CADS genes,CAD1-A subfamily genes are mainly expressed in roots and ovules,CAD1-C subfamily genes are expressed in various tissues of cotton.CRISPR vectors for knocking out CAD1-A and CAD1-C respectively and knocking out these two genes simultaneously were constructed,and the editing efficiency of vectors were detected in hairy roots and cotton calluses,calluses where mutations were detected continue to be cultured into seedlings.We obtained mutants with different degrees of gossypol reduction.In sg A4-1plant with the CAD1-A gene knocked out,the content of gossypol in T₂ seeds decreased by 46.4%compared with R15,and the content of gossypol in leaves did not change significantly compared with R15;In sg C1-2 plant with CAD1-C gene knocked out and sg AC2-1 plant with CAD1-A/CAD1-C genes knocked out,the gossypol content of seed was reduced by64.1%and 47.4%compared with R15,respectively,and the content of gossypol in cotton leaves was also significantly reduced.In addition,the oil and protein content of T₂ generation cottonseeds and the agronomic traits（plant height and fiber quality）of the mutants were analyzed,the results indicated that gossypol reduction had no direct effect on oil and protein content and agronomic traits.3.Through transcriptome analysis,the decrease in gossypol content affected the biosynthesis of other metabolites and signal transduction pathways,including terpene biosynthesis,flavonoids biosynthesis,JA biosynthesis,MAPK signaling pathway,etc.The JA and GA biosynthetic pathway genes were down-regulated,which was consistent with the decrease in JA content and GA content in leaves of mutants.In addition,MYC2 gene in the JA signaling pathway and WRKY1 gene in response to JA were down-regulated,PYR/PYL,Sn RK2 and MPK7 genes in ABA signaling pathway were down-regulated.This suggests that the CADS gene may be involved in the JA and ABA signaling pathways.4.Silencing 6 GhMYC2 genes via VIGS in cotton,we found that the content of gossypol was significantly reduced after silencing MYC2-D09.And the content of gossypol increased significantly after overexpression of MYC2-D09 in hairy roots.Dual-LUC assays showed that MYC2-D09 could activate the promoter activity of CADS genes,but Y1H assay showed that MYC2-D09 did not interact with CADS promoters,indicating that MYC2-D09 may indirectly regulate CADS genes.The same method was used to analyze three GhWRKY1 genes,and it was found that the content of gossypol after silencing or overexpressing WRKY1-D11 gene showed the opposite result to MYC2-D09,and the Dual-LUC assays showed that WRKY1-D11 inhibited the promoter activity of CADS genes,but Y1H showed that WRKY1-D11 did not interact with the CADS promoters,indicating that WRKY1-D11 may indirectly regulate the CADS genes.In this study,we create a new low-gossypol germplasm by knocking out CADS genes via CRISPR/Cas9.The content of gossypol was reduced by 64.1%after knocking out CAD1-A and CAD1-C genes,simultaneously,and the content of gossypol was reduced by 46.4%and 47.4%,respectively,after editing the CAD-A and CAD1-C genes,respectively,which provided new germplasm resources for cotton breeding.In addition,the regulation of CADS genes by MYC2-D09 and WRKY-D11 transcription factors provides a theoretical basis for gossypol biosynthesis regulatory network.