The Application of T-DNA Insertion (Gene Trap) and CRISPR/Cas 9 System in Cotton for Functional Genomic Research

Agrobacterium mediated T-DNA insertion is one of the important strategies to create mutants. In this research, we generated cotton T-DNA insertion mutants with promoter trap system. We use these mutants to isolate T-DNA flanking sequences, analyze GUS expression patterns and the copy number of T-DNAs. Up to now, 2000 + independent T-DNA lines carrying the promoter trap system were created by using Agrobacterium-mediated transformation. Approximately, 50.56% lines had a single copy and each transformants contained 1.83 T-DNA copies in average revealed by southern bolting. More than 1000 flanking sequences from T-DNA left border and right border were identified by using FPNI-PCR. Besides the T-DNA mutation, we also utilized a CRISPR/Cas9 system in cotton for genome editing . An exogenously transformed gene dsRFP and an endogenous gene GhCLA1 were chose as CRISPR/Cas9 knock out targets. For each gene, at least a pair of sgRNAs, driven by a cotton native type Ⅲ RNA polymerase promoter-pGhU6.9, were designed in the coding (or exons) regions. Through Agrobacterium-mediated transformation, the regenerated T0 plants exhibited obvious phenotype for both target genes. For RFP, the regenerated plants had vanished red fluorescence. GhCLA1 targeting plants gained obvious albefaction from embryo to young seedlings stages. Gene mutations were verified with Sanger sequencing and T7E1 enzyme. Large deletions were observed between the paired target sites. Moreover, these albefaction plants only arose in bioallelic mutation of all copies of the target gene. All these results demonstrated that the sgRNA guided Cas9 had a great potential in cotton for genome editing.