Study On The Gene Of CYP71D16 Directed Mutation By CRISPR/Cas9 Technology In Tobacco

Tobacco(Nicotiana tabacum L.)is an important economic crop,and it is widely planted in our country.Tobacco varieties are the main factors affecting the yield and quality of tobacco leaves.At present,the breeding of tobacco varieties in our country is still in the traditional breeding mode,which is mainly based on phenotypic selection.This kind of experience breeding method not only has a long period,but also slow progress of variety improvement,it is difficult to meet the needs of economic development in our country.CRISPR/Cas9 is a new genomic editing technique following the ZFNs and TALENs technique,it has the advantages of convenience,high efficiency,short period and simple structure of vector,etc.,which provides a simple and effective gene mutation technique for improving variety and the quality of tobacco leaves.In order to obtain directional mutants quickly and improve the efficiency of tobacco breeding,CRISPR/Cas9 technique was used to construct a recombinant vector for directed mutation of CYP71D16 gene in tobacco in this study.The vector was used to genetically transform tobacco K326,and the transgenic plants were screened and identified by various methods.The editing technology system of tobacco CRISPR/Cas9 gene was constructed and optimized,and the mutants with directional mutation of tobacco CYP71D16 gene were obtained.The main findings are as follows:1.The CRISPR/Cas9 recombinant vector was constructed: By analyzing the sequence of the tobacco CYP71D16 gene,the targets(recorded as SG39,SG38)were selected on the exons,the targets were connected with the CRISPR/Cas9 vector systems,and two recombinant vectors were constructed.The recombinant vectors used U6 promoter to initiate sgRNA and enhanced CaMV35 promoter to express Cas9 protein,and CaMV35 S promoter was used to express hygromycin resistance gene.The connection of the target sequence and the integrity of the vector were verified by target sequence sequencing and Agrobacterium tumefaciens liquid PCR identification.The results showed that the constructed vector had good integrity,the two recombinant vectors had been introduced into Agrobacterium tumefaciens cells and could be used for further study.2.Optimization of genetic transformation system of recombinant vector: Agrobacterium tumefaciens-mediated leaf disc transformation method was used to carry out genetic transformation experiment,and the technical measures of key links of genetic transformation were optimized.The results showed that:(1)The optimal screening concentration for hygromycin was 20 mg/ L,and the wild-type explants at this concentration were almost non-differentiated and all browned after a period of time.(2)In the activation culture stage of Agrobacterium tumefaciens,the number of single colonies obtained by H1 marking method(parallel and uniform crossing)was the highest.And most of them were distributed independently in a straight line,which could eliminate the contamination of miscellaneous bacteria and ensured the reliability of single colony.(3)In the screening and culture stage of hygromycin,it was found that the contamination rate of infected tobacco leaves could be greatly reduced when 100 mg/L timentin was added.Timetine of higher concentration could completely inhibit the growth of Agrobacterium tumefaciens and other miscellaneous bacteria,but it also caused damage to the leaves.(4)In the dedifferentiation and regeneration phase,it showed that the 6-BA and NAA ratio for 1.0/0.1 was more favorable for callus induction and 0.5/0.1 was more favorable for adventitious bud differentiation.The two hormone combinations could give consideration to both callus induction and adventitious bud differentiation at the same time.(5)In the rooting stage,when the IAA and IBA ratio was 0.2/0.2,the rooting amount was more,the average root length was the longest and reached 3.23 cm,the root was thicker,the leaf color was normal,and the growth potential was better.3.Screening of positive plants and identification of mutagenic effect: PCR method was used to screen and identify hygromycin resistance gene and Cas9 gene of transformed plants.52 positive strains were identified by PCR detection to hygromycin,and 46 positive strains were identified by detection to Cas9 gene.The positive rate was 82.14%.The results showed that 11.54% of the Cas9 genes were lost or silenced in the integration process.In order not to affect the accuracy of the screening results.It was suggested that PCR detection to Cas9 gene should be used to screen positive strains.Moreover,sequencing and RT-qPCR methods were used to identify the gene mutation effect of positive strains.The results of sequencing showed that there were changes of single base and amino acid in the target and vicinity of 39 positive strains,and the editing efficiency was 84.78%.The results of RT-qPCR analysis showed that the relative expression of CYP71D16 gene in 38 positive mutants were lower than that in wild-type plants,and the plant rate of the target gene mutation was 82.61%.The results showed that there were differences between the two methods.The possible reason was that the target gene sequence was changed by sequencing method,and the change of gene expression was identified by RT-qPCR method.A change in gene sequence does not necessarily result in a change in the expression of the target gene,so we can use both methods or only the latter in the identification of mutants.In this study,by using the CRISPR/Cas9 technology to directionally mutate the tobacco CYP71D16 gene,38 mutational materials were obtained and the plant rate of the target gene mutation was 82.61%.The results showed that the mutation efficiency of CRISPR/Cas9 gene editing technology was high.The CRISPR/Cas9 gene editing technology system constructed in this study meet the practical requirements of directed mutation of tobacco gene.