Cloning Of U6 Promoters And Establishment Of CRISPR/Cas9 Mediated Gene Editing System In Eggplant

Eggplant（Solanum melongena L.）is an important worldwide vegetable crop,which is widely cultivated in China and have an important economic value.CRISPR/Cas9 gene editing technology has attracted increasingly attention due to the advantages of simple operation and lower off-target activity.However,the successful application of CRISPR/Cas9 gene editing system has not been reported in eggplant.The U6 promoter is the one of an important element in the CRISPR/Cas9 gene editing system.In the establishment of gene editing system in dicotyledonous plants,U6 promoter was often used to drive sg RNA expression.At present,the corresponding endogenous U6 promoter has been successfully cloned in various plants.However,the same U6 promoter is not applicable to all species,especially those with distant genetic relationships.The U6promoter suitable for eggplant and capable of efficient transcription has not been reported.In this study,we were planning to clone the U6 promoters from the genome of eggplant and screening out the Sm U6 promoters that has higher transcriptional activity.Its were used as an ideal promoters to construct CRISPR/Cas9 gene editing vectors.Therefore,an efficient CRISPR/Cas9 gene editing system was established in eggplant.The main results of this study are listed as follows:1.Seven Sm U6 promoters were cloned successfully and the sequences analysis were carried out.Seven candidate U6 promoters were found in eggplant genome in this study.The candidate U6 promoters were trunked at the 5’end before designed amplification primers.Seven Sm U6 promoters which had been trunked were cloned by high fidelity DNA polymerase and named as Sm U6-1P,Sm U6-2P,Sm U6-3P,Sm U6-4P,Sm U6-5P,Sm U6-6P and Sm U6-7P,with lengths of 415 bps,346 bps,211 bps,437 bps,305 bps,501 bps and581 bps.Analyzed the sequences of seven eggplant U6 promoters and Arabidopsis U6promoter,we found that At U6-P,Sm U6-1P,Sm U6-2P,Sm U6-3P,Sm U6-4P and Sm U6-7P were containing with the TATA box and USE（upstream sequence element）element（5’＿TCCCACATCG＿3’）which were recognized and binded by polⅢRNA polymerase,and the base number between the two components were fixed and about 30 bps.2.Five Sm U6 promoters showed transcriptional activity by tests.Eight GUS fusion expression vectors were constructed in this study,which were Sm U6-1P::GUS,Sm U6-2P::GUS,Sm U6-3P::GUS,Sm U6-4P::GUS,Sm U6-5P::GUS,Sm U6-6P::GUS,Sm U6-7P::GUS and At U6::GUS.These vectors were injected into tobacco leaves by agrobacterium-mediated transformation.The results of GUS chemical staining showed that six kinds of injected tobacco leaves were stained successfully,indicating that five Sm U6promoters could drive the expression of GUS reporter gene and these five Sm U6 promoters were capable of transcriptional activity.Sm U6-1P and Sm U6-7P with deeper staining were selected to construct CRISPR/Cas9 vectors.3.The effectiveness between Sm U6-1P and Sm U6-7P were compared in CRISPR/Cas9 system of eggplant,so as to establish an CRISPR/Cas9 gene editing technology system in eggplant.CRISPR/Cas9 gene editing vectors were constructed using Sm U6-1P/Sm U6-7P/At U6-P to drive Sm WRKY4/Sm WRKY26 sg RNA,which were Sm U6-1P::Sm WRKY4-sg RNA-Cas9,Sm U6-1P::Sm WRKY26-sg RNA-Cas9,Sm U6-7P::Sm WRKY4-sg RNA-Cas9,Sm U6-7P::Sm WRKY26-sg RNA-Cas9,At U6-P::Sm WRKY4-sg RNA-Cas9 and At U6-P::Sm WRKY26-sg RNA-Cas9.T₀plants were obtained by agrobacterium mediated transformation of eggplant cotyledons.The positive rate and mutation rate were detected by two rounds of PCR.A total of 37 regenerated plants were obtained based on Sm U6-1P,including 32 positive plants and 9 mutant plants,with a positive rate of 86%and a editing efficiency of 28%.A total of 18 regenerated plants based on Sm U6-7P were obtained,including 14 positive plants and 3 edited plants,with a positive rate of 78%and a editing efficiency of 21%.A total of 5 regenerated plants based on At U6-P were obtained among which 4 were positive plants.However,no edited plants were detected.The results showed that Sm U6-1P and Sm U6-7P could drive the expression of sg RNA and edit the target genes.In addition,the statistical results showed that the editing efficiency of Sm U6-1P was higher than that of Sm U6-7P.In this study,an effective CRISPR/Cas9 system for eggplant was successfully established,which will provide a technical support for the research of gene function and molecular breeding in eggplant.