Abstract: 【Objective】In order to take advantage of the genome editing technology in cotton molecular breeding, U6 promoters were cloned from cotton variety Xinhai16 (Gossypium barbadense L.) and then a GbU6 promoter was selected with high transcription activity in cotton germ cells (pollen), which will lay an important foundation for cotton molecular breeding.【Method】Two rounds of PCR were adopted to clone accurately the full-length of GbU6 promoter without redundant sequence at 3’ end, five pairs of primers were designed using software Primer Premier 5.0. The first round PCR was carried out to obtain DNA fragments which cover five full-length GbU6 promoters, respectively. After confirming by sequencing, five GbU6 promoters were subcloned precisely into CRISPR/Cas9 genome editing vector by the second round of PCR (transfer PCR). After right sequencing of the transfer PCR products, the necessary element for transcriptional function of 5 kinds of GbU6 promoter was analyzed using DNAMAN software. Then GUS report gene was cloned using pBI101 plasmid as template. After confirming by sequencing, GUS gene was cloned into CRISPR/Cas9 genome editing vectors carrying five GbU6 promoters, respectively, by BbsⅠdigestion and ligation, which create five kinds of fusion expression vectors of GbU6::GUS. DNA fragments of CaMV35S promoter-driven GUS and fiveGbU6::GUS were obtained through High fidelity PCR and then were transferred into cotton pollen using a particle gun. Results were observed using a stereomicroscope after GUS staining. Each transformation was repeated three times. 【Result】Five different GbU6 promoters were cloned after two rounds of PCR and they contain 1 166 bp, 1 119 bp, 1 134 bp, 1 214 bp and 1 176 bp, respectively. Construction of the corresponding five CRISPR/Cas9 genome editing vector with five different GbU6 promoters were done. After sequence comparation of U6 promoter between Arabidopsis and cotton, results showed that cotton U6 promoter contained the conserved -60 USE motif and -30 TATA box and the distance between two elements also was fixed, just like Arabidopsis U6 promoter. The results of transient transformation showed that four cloned GbU6 promoters could drive the expression of GUS gene in cotton pollen which were stained into blue. Among the four promoters, the GbU6-5P::GUS exhibited deeper blue, similar to the CaMV35S promoter.【Conclusion】GbU6 promoters with high-level transcription in cotton pollen were cloned, thus providing an efficient promoter for genome editing technology of CRISPR/Cas9 in cotton.

Key words: cotton;GbU6 promoter, cloning, pollen, transient expression