| CRISPR/Cas9(The Clustered Regularly Interspaced Short Palindromic Repeats-associated Endonuclease 9)is a new gene editing technology that application in 2013.It can modify target genes specifically and has been used for genome editing research in many species due to its easy operation.It is of important value to gene function identification and crop molecular design breeding.U3 or U6 promoters whose transcription initiation sites have been confirmed is one of important component of the CRISPR/Cas9 gene editing system.At present,the cotton genome sequencing has been completed.Several candidate U3 and U6 promoters were identified by sequence analysis in the cotton diploid genome.Whether these promoters can achieve the gene editing function in cottonthrough CRISPR/Cas9 system is not very clear.Firstly CRISPR/Cas9 gene editing vectors were constructed by using Gb U6-1P,Gb U6-7P and Gb U3-2P promoters to drive sg RNA respectively,in which GGB,a negative regulator of drought stress,was used as target sequence.Secondly the function of the gene editing vector was identified in protoplasts of cotton Xinhai 16.The detailed results are as follows:(1)The above CRISPR/Cas9 gene editing vectors were identified by digestion of restriction enzymes(Hind III,Kpn I and Xba I).The results showed that the size of the bands was consistent with the expected design size,indicating that the CRISPR/Cas9 gene editing vectors based on the Gb U3-2P,Gb U6-1P,and Gb U6-7P promoters have all been successfully constructed and are named Gb U6-1P::GGB-sg RNA-Cas9,Gb U3-2P::GGB-sg RNA-Cas9 and Gb U6-7P::GGB-sg RNA-Cas9 respectively.(2)A large number of core fragments of the constructed CRISPR/Cas9 gene editing vector were enriched by PCR method,and then were transferred into protoplasts using the PEG transient transformation method.Genomic DNA of the transformed protoplast was extracted and digestion/PCR method was used to analyze the mutation of cotton GGB gene and the mutations were confirmed by sequencing.Finally,in order to confirm the editing efficiency and authenticity of the CRISPR/Cas9 system,the frequency distribution map of mutations in target gene was mapped according to sequencing results.The results showed that the cotton endogenous target gene sequence is edited successfully by the CRISPR/Cas9 gene editing systems using Gb U3-2P,Gb U6-1P,and Gb U6-7P as promoters.All the mutation types were base substitutions.(Including both conversion and transversion),mutation rates were 21 %,34.5 %,and 26 %,respectively. |
