Establishment Of CRISPR/Cas9 Technology And Its Application On Diospyros Lotus L.

The CRISPR/Cas9 system is an emerging biotechnology in recent years,which facilitate the genome modification.It has been gradually applied to genome editing research of various plants.There were no report on CRISPR/Cas9 in Diospyros up to now.The accumulation of proanthocyanidins in the persimmon fruit pulp is the casue for astringency.ANR plays a key role in the process by converting anthocyanins into epicatechin(precursor of PAs).Carotenoids are important pigments involved in the color formation of persimmon fruits,leaves and other tissues and organs.The PDS gene plays a key role in carotenoids biosynthesis process and can catalyze the conversion of colorless phytoene into colored carotenoids.In this research,the CRISPR/Cas9 technology was used to knock out Nt PDS gene of tobacco,the Dl ANR,Dl PDS gene of Diospyros lotus L.respectively.A binary expression vector containing two targets was constructed and then the genetic transformation was achieved by leaf disc method.Phenotype and sequencing were carried out to evaluate the gene editing performance.The main research results are as follows:1.Vectors PHSE401-GFP and PRGEB32-Gh6.7S-Cas9 was used to construct PHSE-2g R-Nt PDS,p RGEB-2g R-Nt PDS recombinant vectors for the Nt PDS gene editing in tobacco.Transgenic plantlets were regenerated by leaf disc method.About 200-300 bp fragment around the target gene locus was amplified.Sequencing was performed to detect mutations in the target gene of the two regenerated seedlings introduced with p RGEB-2g R-Nt PDS vector.The subsequent positive plants produced an obvious albino phenotype.2.The above gene editing vectors PHSE401-GFP and PRGEB32-Gh6.7S-Cas9 were used to construct the target gene knockout vectors PHSE-2g R-Dl ANR,PHSE-2g R-Dl PDS,p RGEB-2g R-Dl ANR,p RGEB-2g R-Dl PDS for Diospyros lotus.Robust fluorescent signal was detected on the regenerated callus transferred with PHSE401-GFP vector.The Cas9 protein fragment of PRGEB32-Gh6.7S-Cas9 vector was amplified using PCR,but no expected sequence variation of the target locus.A CRISPR/Cas9 system based on fusion vector combining Cas9 and multiple sg RNAs was established to knockout of endogenous genes in tobacco.In addition,CRISPR/Cas9 technology was applied on Diospyros lotus.