Application Of CRISPR/Cas9 Technology In The Production Of Hairy Roots Of Salvia Miltiorrhiza With High Salvianolic Acid

Salvia miltiorrhiza is a Lamiaceae sage plant.It is a traditional Chinese medicine with the functions of promoting blood circulation and removing blood stasis,clearing the meridian and relieving pain,cooling blood and relieving pain.It is often used to treat chest pain,irregular menstruation,cardiovascular and cerebrovascular diseases,and is one of the commonly used bulk medicinal materials in my country.Because of its fast growth rate,small genome,short generation cycle,and the maturity of tissue culture technology and genetic transformation system,it is considered as an ideal medicinal model plant.Salvianolic acid B is an important plant secondary metabolite in Salvia miltiorrhiza.It has strong scavenging ability to free radicals,and has anti-inflammatory,analgesic and antioxidant effects.wide attention.4-hydroxyphenylpyruvate dioxygenase is a bypass gene on the tyrosine synthesis branch in the salvianolic acid biosynthesis pathway,which catalyzes the formation of homogentisic acid.The metabolic accumulation of salvianolic acid plays a regulatory role.In order to meet the growing demand for salvianolic acid compounds in the medicinal market,this study used the sterile seedling of Shannong Danshen No.1 as the test material,and used the CRSIPR/Cas9 gene editing technology to design 6 sg RNAs targeting the Sm HPPD gene,and successfully constructed them.6 corresponding CRISPR/Cas9 knockout vectors.The leaves of Salvia miltiorrhiza were transformed by Agrobacterium rhizogenes ACCC10060,and the hairy root material with the deletion of Sm HPPD gene was obtained by stepwise culture.Using PCR amplification detection,the knockout vectors have been integrated into the hairy roots of Salvia miltiorrhiza.A total of 17 transgenic positive hairy roots were obtained,with a transformation efficiency of 77.27%.Analysis of DNA sequencing results: a total of 7 positive hairy roots were mutated at the corresponding target positions,of which 6 were chimeric mutations and 1 was biallelic mutation.The mutation types included substitutions and deletions,and the editing efficiency was 41.18 %.The expression levels of Sm HPPD gene in the edited lines were detected by q RT-PCR,which were significantly lower than those in the control lines,among which H3-2,H4-1,and H4-2 were the most obvious,which decreased by 86.1%,81.18%,and 86.96%,respectively..The edited strains were further tested by UPLC to observe the changes of phenolic acids and tanshinone compounds.The results showed that the contents of rosmarinic acid and salvianolic acid B compounds in the edited strains were significantly higher than those in the control strains,among which H3-The two strains had the most obvious changes,which were 2.85 times and2.38 times that of the control.There was no significant difference in tanshinones.It can be seen that knocking out the Sm HPPD gene can effectively increase the content of phenolic acid metabolites.In this experiment,CRISPR/Cas9 gene editing technology was used to knock out the bypass gene Sm HPPD for salvianolic acid synthesis.By reducing the expression of this gene,the synthesis metabolism of the competitive bypass was reduced,thereby promoting the accumulation of salvianolic acid compounds and obtaining the deletion of the Sm HPPD gene.hairy root material.The effects of Sm HPPD gene on the synthesis of salvianolic acid compounds were verified,and the content of salvianolic acid compounds was increased.CRISPR/Cas9 gene editing technology was successfully applied to laboratory production of salvia miltiorrhiza hairy roots,which provided a potential method for the improvement of salvia miltiorrhiza varieties.