Cloning And Expression Analysis Of LOX And HPL Genes In Hedychium Coronarium

Hedychium coronarium is a perennial herb which belonging to zingiberaceae.It is popular with people because of their beauty,white color and charming fragrance.Terpenes and their derivatives consist of the main volatile flavor scent.In this paper,we used Hedychium coronarium as the research object,through RT-PCR,real-time quantitative PCR,subcellular localization and GC-MS technologies,the LOX and HPL genes in LOX pathway were cloned and finished the s ubcellular localization experiment.What is more,the expression patterns of LOX and HPL genes were studied in coronarium of different flowering stages,different tissues and different flavors,Which provided some theoretical basis for further elucidating the role of LOX pathway in flower metabolism.The main results obtained in this paper are as follows: 1.The number,phylogenetic relationship,protein structure and classification of LO X family members in Hedychium coronarium transcriptome were analyzed by bioinformatics method.The results show that there are seven protein belonging to LOX family in the Hedychium coronarium.Among them,9-LOX and 13-LOX have 1 and 6 members respectively.The amino acid sequence identity of them was between 39.51 and 68.31%.Their isoelectric points are higher than 6.0 except the HcLO X7 gene which isoelectric point is 5.40.2.The number,phylogenetic relationship,protein structure and classification of CYP74 B / C family members in the P450 protein family were analyzed by bioinformatics method.we found three HPL protein in Hedychium coronarium and the similarity of them between 40.00 and 52.57%.Hc HPL1 is acidic amino acids,and the others are basic amino acids.The isoelectric points of Hc HPL1,Hc HPL2 and Hc HPL3 are 5.84,8.77 and 8.11 respectively.3.Two cDNA full complete sequences named as HcLOX1 and HcHPL1 respectively,were isolated from Hedychium coronarium flower by RT-PCR.The ORF c DNA of HcLOX1 and HcHPL1 contained 2745 bp and 1464 bp,and encoded 915 and 488 amino acids respectively.4.The transient expression vectors of HcLOX1 and Hc HPL1 were constructed.Both of the proteins were fused with GFP protein and expressed transiently in Arabidopsis protoplasts,.The results revealed that Hc LOX1 was located in the chloroplast,while Hc HPL1 was localized in mitochondria.5.The expression levels of HcLOX1 and Hc HPL1 in different tissues of Hedychium coronarium were analyzed by RT-qRCR.The results revealed that HcLOX1 and HcHPL1 were expressed most in the lateral flap of the flowers,and were also expressed in the labial and labialis,while the styles,anthers and filaments,and the vegetative organs leaves and rhizomes were almost no expressed.In the other hand,the expression levels of HcLOX1 and HcHPL1 varied with the development of flowers during the different developmental stages of Hedychium coronarium.HcLOX1 and HcHPL1 were not expressed before the flowers were opened,and the expression of HcLOX1 and Hc HPL1 was increased rapidly at 1741 times and 316 times in Semi-open period.HcLOX1 expression in the flowering period rose to the highest,and then gradually decreased with the flower aging.while HcHPL1 in Semi-open period rose to the highest expression and then declined gradually,however,the HcHPL1 has rised slightly after decline.Last,The expression levels of HcLOX1 and HcHPL1 were the highest in the aroma-type Hedychium coronarium,which was 8.16 and 16 times more than the light fragrance Hedychium gardnerianum.The two genes were both not expressed in the non-fragrant Hedychium coccineum.6.The GC-MS technique was used to analyze the voliate scent of Hedychium coronarium treated with the Lipoxygenase Inhibitor called NDGA.The results showed that contrast to the control flowers,the terpene volatiles,such as linalool,1,8-cineole,ocimene and allo-ocimene declined 56.4%,39.0%,53.9% and 59.3% respectively,and the phenylpropane methyl benzoate decreased 69.6%.7.The divergence expression of Hc HPL1 and Hedychium coronarium Floral related genes were analyzed with Real-time RT-PCR.Compared with the control,NDGA treatment decreased the expression of the gene Hc BSMT2,which regulated aromatic compounds in phenylpropane pathways dropped 36.9%.Some of the functional genes involved in the biosynthesis of terpene,such as HcTPS1,HcTPS3,HcTPS5 and HcTPS8 declined 62.7%,68.4%,25.8% and 21.8% respectively.The downstream gene HcHPL1 of the LOX pathway also results in down-regulation,dropped 34.5%.