Effects Of Biochar On Carotenoid Metabolism And Over-Expression LCY-b Gene In Flue-Cured Tobacco

To identify the effects of biochar on leaf carotenoids of tobacco,the tobacco variety(Nicotiana tabacum L,K326)was planted in Guangchang county.The carotenoid contents and the neutral aroma components were compared as well as the expression of several key genes related to carotenoid metabolism in K326 after applying different amounts biochar.on this basis,using homology cloning strategy,the full-length c DNA sequence of LCY-b was cloned from tobacco variety K326.Then this gene was overexpressed in tobacco by agrobacterium-mediated transformation.The carotenoid components and the aroma compounds of the transgenic plants were also determined.Hoping to reveal the function of lycopene beta-cyclase(LCY-b)gene in the growth and development of tobacco plants,especially the influence on aroma substance metabolism.The main finding are as follows:1.With the development of growth period,the expression of the genes related to carotenoids synthesis reduced gradually after topping,while the expression of the genes related to carotenoids degradation increased.The results were consistent with the finding that the carotenoid content decreased gradually after topping.At the later stages of middle leaf maturing,biochar application increased the carotenoid accumulation in leaves,the carotenoid accumulation keep at a higher level in leaves treated by biochar at the rate of 300 kg/ha.The analysis of gene expression displayed that the expression of the genes related to carotenoid metabolism were higher in the treatment of applying 300 and 600 kg biochar per hectare.After being cured,the contents of the degradation products of carotenoids were more in the treatment of applying 300 kg biochar per hectare.These results showed that the right amount of biochar enhanced the enzyme activity related to carotenoid metabolism.Besides,the metabolism of tobacco carotenoids and the contents of the degradation products of carotenoids were promoted by applying moderate biochar.2.Using homology cloning strategy,the full-length c DNA sequence of LCY-b was cloned from tobacco variety K326.And carried on the bioinformatics analysis.Sequences analysis showed that tobacco LCY-b contained 1503-bp open reading frame(ORF)and coded 500 amino acid residues,the calculated molecular masses was 56.05 KDa with the theoretical isoelectric point(p I)of 6.68.The secondary structure prediction result showed that they were mainly made up by alpha helix and random coil.3.The overexpression vectors was constructed using in-fusion methods.Then the vectors were transplanted to tobacco using agrobacterium mediated method to achieve LCY-b gene overexpression in tobacco.Molecular testing proved LCY-b gene had achieved the integration and expression in tobacco.The expression of LCY-b gene was significantly higher in some of the transgenic positive plants than that of K326 untransformed control.Correspondingly,the proportion of beta-carotene,neoxanthin and violaxanthin were increased.While the the proportion of lutein were reduced.The analysis of aroma susbstance in the cured sample also display that the amounts of beta-carotene degradation products was significantly higher in transgenic positive plants than that of K326 untransformed control.But the lutein degradation products was equivalent to or lower than that of K326 control.These results showed that LCY-b could change the proportion of carotenoid components and affect the content of aroma components in cured samples.