Expression Characteristics And Regulation Of CBT-diol Synthase Gene(NtCYP71D16) Of Tobacco

Cytochrome P450 hydroxylase(NtCYP71D16)catalyzes the dehydrogenation of CBT-ol to produce CBT-diol,which is a key enzyme gene for the biosynthesis of Cembranoids.Chemical composition has an important impact.In this paper,the Nt NtCYP71D16 gene of tobacco cultivar K326 was cloned,and the expression characteristics of the gene and the response to temperature and Me JA were analyzed by RT-PCR and Promoter-GUS infusion.The results were compared.(NtCYP71D16 and NtCYC),and the expression of NtCYP71D16 gene in K326 was realized by RNAi technique.The expression of NtCYP71D16 and NtCYC in the RNAi transgenic plants was studied.The chemical composition,the resistance and the aroma components of the flue-cured tobacco leaves were compared and analyzed.The results of this study laid a theoretical foundation for the application of NtCYP71D16 gene in tobacco leaf chemical regulation and its resistance and quality improvement.The main findings are as follows:1、The NtCYP71D16 gene of flue-cured tobacco K326 was cloned by homologous cloning technique.Through the nucleotide sequence alignment,we speculated that the genes we were all tobacco NtCYP71D16 gene,temporarily named NtCYP71D16,NtCYP71D16v2,NtCYP71D16v3.The homology of NtCYP71D51v1 and NtCYP71D51v2 and NtCYP71D51v3 were more than 80%.The sequence identity of NtCYP71D16v2 and NtCYP71D16v3 was 93.9%,and the consistency of NtCYP71D16v2 and NtCYP71D16v3 was 96.1%.The protein domain of this gene was analyzed.It was found that all three proteins contained the conserved domain of P450 protein family,which indicated that the cloned gene was tobacco P450 family gene.2、(1)RT-PCR technique was used to analyze the expression of NtCYP71D16 gene in different tissues of K326 root,stem,leaf,glandular hair,leaves without glandular hair and flowers.The results showed that NtCYP71D16 gene of expression level in the leaves was higher than that in the leaves,and the expression level in the leaves of the glandular hair was lower and the expression level was lower in the seeds,stems and roots.(2)The promoter activity of NtCYP71D16 gene was analyzed by Promotor-GUS infusion technique.The CYP-GUS transgenic plant GUS histochemical staining showed that NtCYP71D16 gene was mainly expressed in stem,leaf and flower glandular hair expression.(3)CYP-GUS transgenic plants were treated at 4 ℃,28 ℃ and 42 ℃ respectively.GUS activity test showed that high temperature and low temperature could induce CYP gene expression.(4)After spraying the 0.8% Me JA solution by CYP-GUS transgenic plants,there was no obvious blue color in the glandular hair after GUS staining,which indicated that Me JA could inhibit the expression of NtCYP71D16 gene.3、To characterize the formation mechanism of tobacco trichome exudates,five representative flue-cured tobacco cultivars were studied in trichome morphology,leaf surface chemical components,and expression patterns of genes related to trichome exudates biosynthesis using super depth of field microscopy,GC-MS analysis,and real-time fluorescent quantitative PCR techniques,respectively.The results indicated that(1)two leaf trichome types of flue-cured tobacco were identified,including glandular trichomes and non-glandular trichomes.The proportion of each type was different in these tobacco cultivars,such as glandular trichomes were more abundant and dense in “Cuibi1” and “Yuyan11” compared to other cultivars,while non-glandular trichomes were most abundant in “Zhongyan 100”.(2)Cembranoids are main compounds in tobacco trichome exudates.Among these cultivars,cembranoids were most abundant in “K326”.As the main trichome exudates of oriental tobacco,labdanoids were rarely detected in these flue-cured tobacco cultivars except for “yuyan11”.(3)Expression pattern analysis showed that the expression level of cembranoid biosynthetic related genes(NtCYC and NtCYP71D16)was highly in “K326”.Meanwhile,labdanoids biosynthetic related genes(NtCPS2 and Nt ABS)expressed strongly in “yuyan 11”.These results provide new perspectives for tobacco breeding and the metabolic engineering of trichome exudates.4、Inhibitory expression of NtCYP71D16 gene in K326: The expression vector p K7GWIWG2(II)-NtCYP71D16 of NtCYP71D16 gene was constructed by RNAi technique.K326 was transformed by Agrobacterium tumefaciens-mediated transformation,and the positive plant was gained through the selective MS medium that concentration was 30mg/l,The results showed that the expression of NtCYP71D16 gene was significantly inhibited by molecular detection.The results showed that the content of CBT-ol in the two lines was higher than that of K326,and the content of CBT-diol was lower than that of K326.The aphid treatment of transgenic plants showed that CYP-RNAi transgenic plants had significant resistance to the results showed that the degradation products of cembranoids were significantly lower than those of control K326 in the aroma analysis of transgenic plants.