Function Analysis Of GhAGL1 In Cotton Fiber Development

Cotton（Gossypium L.）is one of the world’s most important economic crops and occupies an important position in our national economy.Cotton fibers develop from single cell protuberances in the ovule epidermis,which act as an important model for exploring cell elongation and cell wall formation.The MADS-box gene family plays an important role in floral organ development and signal transduction in plants,but its role in cotton fiber development has been rarely reported.In this study,Gh AGL1,a gene preferentially expressed in ovules at the fiber initiation,was screened from Gossypium hirsutum TM-1expression profile data.The Gh AGL1 mutant was created by using CRISPR/Cas9technology.The mutant phenotype was investigated and the role of the gene in cotton fiber development was analyzed.The main results were as follows:1.Analysis of Gossypium hirsutum TM-1 expression profile data showed that Gh AGL1 was mainly expressed in ovules,with the highest expression at 0 DPA（Day post anthesis）,after which it started to gradually decrease,and the expression level of GHAGL1in D subgenome was higher than that in A subgenome.By comparing the nucleotide sequence with the amino acid sequence of Gh AGL1,it was found that Gh AGL1 had a distinct MADS structural domain and K-box domain,which meaned that Gh AGL1belonged to the transcription factor of MIKC type MADS-box gene of AG subfamily,which related to the growth and development of floral organs.2.All single strains of the T₁,T₂,and T₃ generations of the Gh AGL1 knockout lines were examined for editing using Hi-TOM sequencing technology,and the results showed that each single strain had at least one target edited,theedited type was stably inherited to future generations,and two copies of Gh AGL1 gene could be knocked out basically.The Gh AGL1 knockout lines was subjected to continuous self-crossing,and the phenotypes of plants from T₁ and T₃ generations were examined,and the knockout lines were found to have inhibited fiber initiation,significantly shorter mature fibers and poorer fiber quality compared to the control strain（Jin668）.3.We screened interacting proteins in a lab-built library of fiber initiation-associated transcription factors by using yeast two-hybrid,with Gh AGL1 as a bait protein,followed by in vivo interaction validation,which resulted in the interaction of Gh AGL1 with four TFs,including Gh AGL9,Gh AGL4（a）,Gh AGL4（b）from MADS family and Gh AGL6（b）from MICK-type MADS family.Analysis of the expression patterns of genes encoding interacting proteins revealed that these four genes were similar to Gh AGL1 expressionGhAGL1 patterns and all were preferentially expressed during the fiber initiation stage..4.To investigate the regulatory network on the downstream genes of Gh AGL1,the transcriptome of 0 DPA ovules from its three knockout lines and the control strain（Jin668）were sequenced and analyzed,and a total of 174 differential genes（DEGs）,which were mainly enriched in the regulatory pathways of cell wall,phenylpropanoid biosynthesis as well as glycosphingolipid biosynthesis-globo and isoglobo series,including transcription factors in the hormone signaling pathways of gibberellin,abscisic acid and salicylic acid,such as Gh MYB101,Gh DPBF2,Gh WRKY70,along with Gh WRI1 as an ethylene response factor,Gh MYB52 as an abscisic acid and salt stress response factor.