Preliminary Functional Studies On The Upland Cotton 4-coumarate Coenzyme A Ligase Genes Gh4CL20/20A And Gh4CL7/25

【Object】The 4-coumarate coenzyme A ligase(4CL)is involved in the phenylpropane metabolic pathway,providing precursors for many secondary metabolites in plants and regulating the production of metabolites such as lignin and flavonoids.4CL genes have important roles in responding to biotic and abiotic stresses,influencing plant fertility,and regulating environmental adaptations.So far,4CL genes have been extensively studied in several species,but there are relatively few relevant reports of such studies in cotton.In this study,we identified and explored the function of 4CL gene in land cotton and obtained Gh4CL20/20 A and Gh4CL7/25 gene edited plants from land cotton.In addition,this study was conducted to investigate the mechanism of 4CL gene regulation of cotton pollen development based on the mutant material,and to provide a theoretical basis for cotton molecular breeding.【Methods】Gh4CL20/20 A and Gh4CL7/25 were identified as the key genes in this study;CRISPR/Cas9 gene editing vectors were constructed and genetically transformed to obtain mutant plants,and their phenotypic traits were identified and analyzed.We used pollen staining,paraffin tissue sectioning and other methods to analyze the morphological changes of different anthers and pollen;measured the metabolite content in the anthers of wild type and mutant at different times,and analyzed the changes of metabolite content during the development of anthers;used RNA-seq to analyze the differences in transcript level during the development of anthers,and performed GO and KEGG analysis on the differential genes to preliminarily study the molecular mechanism of Gh4 CL gene regulation of fertility.【Results】1.35 4CL genes were identified in the land cotton genome database(ZJU,version 2.1),mainly including 4CL(8)and 4CL-like(27).4CL gene expression patterns revealed that Gh4CL20/20 A was significantly expressed in petals and anthers;Gh4CL7/25 and Gh4CL12/30 were significantly expressed in stems,roots and Gh4CL7/25 and Gh4CL12/30 were significantly expressed in stem,root and pistil;Gh4CL6/24 was not significantly expressed in different parts.Gh4CL20/20 A and Gh4CL7/25 were used as the key genes in this study.Gh4CL20/20 A and Gh4CL7/12 gene edited transgenic plants were obtained by Agrobacterium-mediated genetic transformation method in cotton.2.CR-Gh4CL20/20 A gene edited plants had petal color changed from yellowish to white,anther development was abnormal,pollen could not be dispersed normally,and the plants were male sterile.Pollen grain activity staining study revealed that WT pollen grains had normal morphology,high pollen number and normal activity;CR-Gh4CL20/20 A pollen grains had significantly reduced number,abnormal morphology and decreased activity.Paraffin sections of anthers showed that WT plants formed relatively complete and full round pollen grains with uniform spines on the surface of pollen wall;CR-Gh4CL20/20 A plants showed a crescent-shaped collapsed pollen grain shape with serious defects in pollen wall structure,and although some spines were formed,their distribution was uneven and pollen was aborted.Transcriptome sequencing(RNA-seq)analysis of 8628 and 2175 differentially expressed genes from the anther tetrad stage and maturation stage,GO enrichment and KEGG pathway results indicated that the differential genes were mainly involved in pollen-pistil interactions,pollen recognition and flavonoid biosynthesis pathways;Gh CHS,Gh CHI,Gh F3 H and The expression of Gh CHS,Gh CHI,Gh F3 H and Gh FLS genes in flavonoid biosynthesis was reduced in CR-Gh4CL20/20 A plants,and the anther flavonoid content was significantly reduced.These results suggest that Gh4CL20/20 A is involved in the regulation of flavonoid biosynthesis process,which in turn affects cotton fertility.3.CR-Gh4CL7/25 gene edited plants were cultured in artificial climate chamber environment and showed normal fertile performance,while CR-Gh4CL7/25 gene edited plants were male sterile under high temperature environment.Flowers at different developmental stages were placed in a high temperature environment,and it was found that pollen growth and development were more susceptible to high temperature during the tetrad period compared to the monodiploid period and the mature period.In vitro pollen germination experiments under high temperature stress showed that the germination rate of both CR-Gh4CL7/25 and WT pollen decreased,with a sharp decrease in CR-Gh4CL7/25 pollen germination rate.The histological observation of anther paraffin sections showed that the pollen grains of CR-Gh4CL7/25 plants under high temperature stress matured early and the pollen walls showed collapsed structures compared with WT.The lignin content assay experiment revealed that the lignin content of CR-Gh4CL7/25 plants was significantly reduced compared with WT.The expression of lignin synthesis-related genes showed that the expression of lignin synthesis-related genes was down-regulated in CR-Gh4CL7/25 plants.Taken together,the Gh4CL7/25 gene regulates plant lignin biosynthesis and enhances the sensitivity of pollen grains to high temperature stress.【Results】In this study,Gh4CL20/20 A and Gh4CL7/25 were found to be closely related to the fertility of cotton;two transgenic plants,CR-Gh4CL20/20 A and CR-Gh4CL7/25,were obtained.The Gh4CL20/20 A gene is involved in the flavonoid biosynthesis pathway,which directly causes male sterility in the plants;the Gh4CL7/25 gene regulates lignin biosynthesis and causes male sterility in the plants under high temperature stress.