Analysis of mutants developed by CRISPR/Cas9-based GhbHLH71 gene editing in cotton.

The basic/helix-loop-helix (bHLH) transcription factors play important regulatory roles in plant growth and development, secondary metabolism, signal transduction, and stress responses. The cDNA of cotton GhbHLH71 gene is 996 bp in length, encoding 331 amino acid residues. Protein sequence of GhbHLH71 contains a conserved bHLH structural domain, which is a member of the bHLH transcription factor family. The qRT-PCR showed that GhbHLH71 gene was relatively highly expressed at 3--12 DPA (days post anthesis, DPA) at rapid elongation stage in cotton fiber, implying that it mainly played a role in cotton fiber development. A CRISPR/Cas9 gene editing vector of GhbHLH71 gene was constructed, and then was transferred into upland cotton (Gossypium hirsutum L.). Six T0 mutant plants were obtained after PCR detection of Cas9 gene and mutation detection of target loci. The phenotypic traits of different T1 GhbHLH71 gene mutant plants were not significantly different from the control during cotton growth and development, but the fiber length of T1 mutants was significantly shorter compared with the control, and T2 generation mutant lines stably inherited the shortened fiber phenotype of T1 generation lines. The shortened fiber lengths of the 4# and 8# mutant lines in two consecutive generations were more than 20% shorter compared with the control, indicating that the mutation of GhbHLH71 gene mainly affected the elongation of cotton fiber cells. This study provides some insight into the biological functions of bHLH transcription factors in cotton and the molecular mechanism of cotton fiber development. [ABSTRACT FROM AUTHOR]