Effects Of Tobacco BBL Gene On Tobacco Growth And Nicotine Synthesi

Berberine bridging enzyme（BBL）plays a key catalytic role in the last step of nicotinic biosynthesis.In order to study the function of TOBACCO BBL gene and create new germplasm,this study selected three target genes BBLa,BBLc and BBLd from small gene family BBL gene family,and isolated the full-length c DNA of BBLa,BBLc and BBLd from tobacco by PCR.The overexpression vectors PSH-CAM-BBLa,PSH-CAM-BBLc and PSH-CAM-BBLd were constructed respectively.Then,according to the CDS sequence of BBL gene published on NCBI,targets were designed on its exons,Oligo dimer was synthesized and connected to CRISPR vector,and plasmid was extracted from escherichia coli to the competent state for sequencing.The plasmid that the sequencing results met expectations was cut with endonucase and then reconnected with T4 ligase.The CRISPR/Cas9 knockout vector was successfully constructed.Agrobacterium tumefaciens infection method was used to transform tobacco.In Hygromycin B screening medium with concentration higher than 10 mg/L,the differentiation of explants was inhibited,and the effect was better when the concentration was 5 mg/L.42 overexpressed positive strains were obtained by GUS staining and PCR,with a positive rate of 35%.Twenty-one strains were knockout positive,with a positive rate of 23.3%.Phenotypic observation showed that the plant height and leaf area of overexpressed T₀ generation increased to varying degrees,and there was a certain degree of variation,while the change of knockout plants was not obvious.RT-q PCR analysis showed that the expression of BBL in the leaves of overexpressed mutant was significantly increased.The expression of BBL was significantly reduced in the knockout plants,indicating that the vector had been successfully introduced into tobacco plants.The nicotine content in positive tobacco leaves was determined by HPLC,and it was found that the nicotine content in overexpressed tobacco leaves increased in different degrees,especially in BBLc overexpressed tobacco leaves.And knock out of nicotine content reduced in different degrees,including BBLa knockout plants fall effect is most obvious,show that BBL genes involved in the synthesis of nicotine,for the subsequent research of BBL mechanism as well as the fully proven pyridine ring and a pyrrolidine ring combination provides reference,for breeding,and do not contain nicotine or low nicotine content in tobacco new strains to lay the foundation.