Over-Expression Of Cembratrien-Ol Synthetase Gene NtCYC In Tobacco

Leaf surface chemical components of tobacoo is closely related to its resistance and leaf quality.To regulate the content and composition of tobacco leaf chemical components,this paper cembratrien-ol synthetase gene NTCYC,and construct overexpression vector p K7WG2-CYC of constitutive plant and overexpression vector p CAMBIA1391-CYP-CYC specifically express in tobacco glandular hairs respectively,using the method of Ti plasmid-mediated to transform tobacco,getting CYC gene constitutive overexpressed transgenic tobacco plant and CYC gene overexpressed transgenic tobacco plant specifically express in tobacco glandular hairs via PCR and Southern blot,q PCR,Northern blot detecting,and the transgenic tobacco plants were analyzed morphological and leaf surface chemical composition.The foundation for influence of the leaf surface chemical composition and resistance of and leaf quality of the overexpression of gene CYC.The main findings are as follows: CYC gene was cloned and analyzed.the primers were designed according to the tobacco sequence in NCBI,using c DNA as template for PCR amplification,the c DNA of the CYC ORF sequence of Nicotiana tabacum K326 of CBTs family was obtained.Through BLAST comparison,we found that and the sequence homology between the nucleotide sequence and the cyclase m RNA of Nicotiana Sylvestris was as high as 99%.CYC gene was constitutively overexpressed in tobacco: the plant expression vector p K7WG2-CYC was constructed.Then Nicotinan tabacum K326 were transformed with the gene via Agrobacterium-mediated transformation,Finally,68 lines of regeneration plant were obtained.The DNA of transgenic plants were extracted,and the PCR detecting indicating that CYC gene was successfully integrated into tobacoo.The q PCR results shows that the expression quantity of a total of 51 is higher than the wild type K326 in 68 regeneration plant.Among them,the expression of CYC can be 40 times more than the controls.CYC gene was overexpressed specifically in tobacco glandular hairs.CYC gene was cloned to glandular hairs specific expression vector p CAMBIA1391-CYP,and the glandular hairs specific expression vector p CAMBIA1391-CYP-CYC was constructed.Then Nicotinan tabacium K326 were transformed with the gene via Agrobacterium-mediated transformation,Finally,7 lines of regeneration plant were obtained.The DNA of transgenic plants were extracted,and the PCR detecting indicating that CYC gene was successfully integrated into tobacoo.six lines were taken to detected via Southern blot,the results showed that the genome DNA of four lines had been successfully inserted CYC gene,including three insertion site is likely to be the same.The q PCR result showed that the expression quantity of 5 lines is higher than the wild type K326 among the 6 lines,could reach 74.8 times in the Maximum.All the 6 lines were ditected purpose band via Northern-blot.Analysis of transgenic plants.The morphological analysis conducted of transgenic plants CYC gene constitutive overexpressed transgenic tobacco plant and CYC gene overexpressed transgenic tobacco plant specifically express in tobacco glandular hairs.Most CYC gene constitutive overexpressed transgenic tobacco plant shows no significant differences compared with the controls,however,the phenotype of part lines changed a lot,appearing the phenomenon of stem bending and plant short.CYC gene overexpressed transgenic tobacco plant specifically express in tobacco glandular hairs shows no obvious change in the phenotype.The leaf surface chemical composition of transgenic plants.The chemical composition of transgenic plants was detected using GC/MS,the results showed that the constitutive overexpression of CYC gene increased the content of cembratrien-ol and cembratrien-diol,and the total of cembratriendiol group.And the content of the sugar easter and Alkanes group shows no obvious change,which shows that the overexpression of CYC had little effect on the content of the sugar easter and Alkanes group.By GC/MS,the leaf surface chemical composition of transgenic plants was analyzed.The results showed that CBT-ol content of the leaf surface chemical composition in transgenic lines C-14,C-20,C-53,C-38 were significantly higher than the control,among them,line C-20 was the highest,which can reach five times around,and line C-7 and line C-40 were lower than that of the control.the CBT-diol content of the leaf surface chemical composition in the transgenic lines C-14,C-53 and C-38 were significantly higher than that of the control.Among them,the C-14 was the highest,which can reach three times,which showed that CYC gene overexpression can benefit to improve the leaf surface chemical composition content CBT-ol and CBT-diol and have no obvious effect.accumulation of sugar esters and alkane compounds.