Cloning And Functional Analysis Of Tobacco PXG And HPL Gene

Tobacco is an important economic crop,and its yield and quality are affected by many factors including genetic factors,soil factors,biotic and abiotic stresses such as topping,pests,droughts,and low temperatures.The lipoxygenase pathway plays an important role in plant growth and development.Hydroperoxide lyase(HPL)and peroxygenase(PXG)are two important enzymes in this pathway.The former catalyzes the peroxide process of fatty acid to form volatile short-chain aldehydes and oxyacids,which are components of plant aroma.The latter catalyzes the formation of epoxy-hydroxy fatty acids,which have been shown to have antibacterial activity.Aroma is an important indicator of tobacco quality.Disease resistance is a guarantee of tobacco production.It is unclear whether PXG and HPL can be used to improve tobacco quality and resistance.Some work had been done and the main results are following.1.The HPL and PXG genes were cloned from tobacco and analyzed by bioinformatics.They were found to have high homology with other species,suggesting that NtHPL and NtPXG had been obtained.The promoter sequences of NtHPL and NtPXG were also found on NCBI,and many cis-acting elements respond to hormones and stress treatments,such as drought,mechanical damage,abscisic acid,indicating that expression of NtHPL and NtPXG may be regulated by a variety of factors.2.Tissue-specific expression analysis of NtHPL and NtPXG showed that they were expressed in the roots,stems and leaves.The former was mainly expressed in leaves,and the latter was mainly expressed in roots.It was also showed that NtHPL and NtPXG were all expressed in tobacco at different development stages.NtHPL was mainly expressed in the seedling stage,and NtPXG was mainly expressed in the mature stage.NtHPL was found to respond to MeJA and mechanical damage treatment.NtPXG responded to MeJA,SA,ABA and other hormone treatments.3.The overexpression vectors and silencing expression vectors of NtHPL and NtPXG were constructed,and then were transfered to tobacco.The transgenic tobacco plants were obtained and identified from the DNA level and RNA level.The expression of NtHPL in the transgenic tobacco plants with NtHPL overexpression vector was 3.6 times compared with the control plants.The expression of NtPXG in the transgenic tobacco plants with NtPXG overexpression vector was 12.3 times compared with the control plants.It indicated that the vectors have been successfully transfered into tobacco plants,and the expression vectors work well.(4)Fungal and TMV infection were conducted.After fungal infection,the leaves of the control group turned yellow,the transgenic plant with the PXG overexpressing plasmid showed little change.After TMV infection,the expression levels of TMV and?-1,4-glucanase were detected by qPCR.Compared with the control,the expression of TMV in the transgenic plant was about 0.6 times that of the control,and the expression level of-1,4-glucanase was higher than that of the control,which suggest that PXG may be involved in response to TMV.