Targeted Knockout Of Nicotine Demethylase Genes And Created Low Harmful Tobacco Material

Tobacco is an important economic crop,it is a solanaceous plant in flower.Compared with other plants,tobacco has higher alkaloid content.Usually,the highest content of alkaloids in tobacco is nicotine.Nicotine can stimulate the nervous system.At the same time,it can help refresh the mind,it is also an important quality factors of cultivated tobacco.Nornicotine can be produced under the catalysis of nicotine demethylation enzyme(NND),nornicotine is a toxic component in tobacco which can cause cancer in laboratory animals,and it also has a direct negative impact on human health.Nornicotine accounts for the percentage of nicotine and nornicotine is called nicotine conversion in tobacco alkaloids.The nicotine conversion of cultivated tobaccos is relatively stable in interspecific differences,but the difference is significant between species,and this difference is mainly caused by the different genes.The genes encoding nicotine demethylation enzyme mainly include CYP82E4,CYP82E5v2 and CYP82E10 in cultivated tobaccos.In this study,the cultivated tobaccos of burley tobacco TN90,hongda and K326 were selected as experimental material.We knocked out the nicotinic demethylation genes to create the quality tobacco material with lower nicotine conversion by the technology of CRISPR/Cas9.The main results of this paper are as follows:(1)We cloned the exon 1 of CYP82E4,CYP82E5v2 and CYP82E10 in cultivated tobacco burley tobacco TN90,hongda and K326 respectively,and performed bioinformatics analysis.The sequencing results shows that the sequences of exon 1 on this three kinds of cultivated tobacco are all 939 bp,and the sequences of the same nicotine demethylation enzyme gene are identical in different cultivated varieties.Bioinformatics analysis shows that the exon 1 of this three genes are all have the highly conservative region of P450 enzyme,and we got same conclusion on region associated with substrate recognition.(2)We designed the knockout target sites on the exon 1 of the nicotine demethylation gene.Each gene was designed for 4 target sites,and there are 2 identical target sites among the three genes.Therefore,there are a total of 14 knockout target sites.We designed primers according to the target sites,we constructed the knockout vectors of nicotine demethylation gene of CYP82E4,CYP82E5v2 and CYP82E10 and the identical knockout vectors of the three genes.Through agrobacterium mediated transformation,we transformed the knockout vector into cultivated tobacco burley tobacco TN90,hongda and K326.We got the T0 positive transgenic seedlings according to resistance screening,and we got seeds by selfing and bagging.We sowed the seeds on MS culture media to screen and obtain T1 positive transgenic seedlings.Through the sequencing of homologous exon 1 of nicotine demethylation gene in T0 positive transgenic seedlings or T1 positive transgenic seedlings,we screened and obtain the knocked out plants.We got 2 CYP82E4 plants,6 CYP82E4v2 plants and 5 CYP82E4 plants among the homozygous knockout plants of T1 generation in burley tobacco,and 5 CYP82E4 plants,2 CYP82E4v2 plants and 1 CYP82E4 plant among the knockout plants of T0 generation in hongda,which included 1double knockout plant of CYP82E4 and CYP82E10,and we also got 3 CYP82E4 plants,4 CYP82E5v2 plants and 1 CYP82E10 plant among the knockout plants of T0 generation in K326.In addition,we also got 6 homozygous knockout plants in T2 generation by self crossing homozygous knockout plants of CYP82E4 gene in T1 generation.(3)We measured the agronomic traits of plants of wild type and the burley tobacco's homozygous knockout plants in T1/T2 generation which had been transplanted 62 days,the results shows that the growth status of the knockout lines was consistent with that of the wild type burley tobacco at maturity,and the absence of nicotine demethylation gene had no negative effect on tobacco growth,even the stem girth and leaf area of plants of cyp82e4?cyp82e5v2 are significantly higher than that of wild type.We dealt the detached leaves of the burley tobacco's knockout plants of T1 generation and that of wild type with concentration of 2% ethephon to make them yellow,simulating ageing process of tobacco leaf.Determined the content of main alkaloids of treated leaves and calculated nicotine conversion using GC-MS/MS method.The result shows that nicotine conversion of burley tobacco TN90 was 5.28%,while the nicotine conversion of cyp82e4,cyp82e5v2 and cyp82e10 were 0.67%,1.15% and 2.45%,respectively,and the nicotine conversion of cyp82e4 was significantly lower than that of control group.All these results show that we have obtained a high quality burley tobacco material which has relatively low harmfulness.