Molecular Cloning And Characterization Of Squalene Synthase Gene And Analysis Of Essential Oils From Different Organs Of Matricaria Recutita

Chamomile (Matricaria recutita L.) is a kind of annual herb from Genus Matricaria in Asteraceae,, which have anti-inflammatory activity, anti-bacterial activity, anti-viral activity and anti-epileptic activity. Chamazulene, bisabolol and other sesquiterpene are the main pharmaceutically active components of Chamomile. As we know, squalene can enhance our immunity activity, heart function activity, anti-tumor activity and anti-fatigue activity, which also is the biosynthetic precursor of triterpenoid saponins, steroids and sterols, etc. Through the role of squalene synthase (SQS), plants can produce squalene by farnesyl pyrophosphate (FPP). The biosynthesis of sesquiterpene and squalene are the two important metabolic pathway of FPP. The activity of squalene synthase can affect the biosynthesis of monoterpenes, sesquiterpene, diterpene and other product from isoprenoid pathway, while the largest influence is the biosynthesis of sesquiterpenes. In this study, we cloned the full-length cDNA of squalene synthase for the first time. The activity detected experiments in vitro show that our purified protein SQS have the squalene synthase activity. Studies have shown that by reducing the activity of SQS can increase the synthesis of non-steroidal substances (including sesquiterpenes). The main contents and results are as follows:1. In our study, we have cloned the full-length cDNA of SQS gene from chamomile for the first time by degenerate PCR and RACE, and we analysis the structure of SQS protein through bioinformatic method.2. Meanwhile, we completed the prokaryotic expression of chamomile SQS gene in vitro, and optimized the protein expression conditions. We obtained a 47.6 KDa protein. Experiment in vitro enzyme activity was detected by GC-MS using FPP substrate. The results showed that the protein have the activity of squalene synthase.3. We have detected the expression of SQS from radial corolla, disk corolla, stem and leave by qRT-PCR in Matricaria recutita, the results show that the expression of squalene synthase in the radial corolla, leaf, stems and radial corolla is reduced in turn. SQS in disk corolla is the highest, which is 11 times higher than radial corolla. Second, the expression of SQS from leave was 3.2 times higher than radial corolla, while stems have little expression of SQS, is about 0.7 times than radial corolla.4. We also extracted the chamomile essential oils from radial corolla, disk corolla, stem and leave by hexane. The composition and content of volatile substances was detected by GC-MS methods. The results showed that the kinds of essential oil compositions we identified from stem, leaf, radial corolla and disk corolla were respectively 43.46,66 and 68. The composition and the content of volatile oil was very different in Matricaria recutita different organs. The volatile oil components of Chamomile disk corolla mainly include:Bisabolene(6.75%),7-methoxycoumar (11.01%), a-bisabolol oxide A(12.9%) and Santonin 34.90%, The volatile oil components of leaves mainly include:a-farnesene(5.52%),7-methoxycoumar (34.72%) and Santonin (37.18%), The volatile oil components of radial corolla mainly include:7-methoxycoumarin (64.86%) and Tetracosane(6.19%), The volatile oil components of stems mainly include:Elixene(3.55%) and 7-methoxycoumarin (24.90%). The proportion of relative content of essential oils from chamomile disk corolla, radial corolla, leave and stem was 2.31:1.66:1.60:1. The relative percentage of sesquiterpene from each organ are as follows:disk corolla(25.56%), stems(21.8%), leaves(15.31%), radial corolla(2.43%). We found that arious organs contain a lot of both 7-methoxycoumarin and Santonin. the contents of 7-methoxycoumar in disk corolla, leaves, radial corolla and stems were:11.01%,34.72%,64.86% and 24.90%; the contents of santonin in disk corolla, leaves, radial corolla and stems were:34.90%, 37.18%,2.24% and 36.92%. Bisabolene(6.75%) and a-bisabolol oxide A(12.9%) are mainly in the disk corolla, and in radial corolla also contain little amounts of a-bisabolol oxide A and B, while other organs do not contain any bisabolol and its oxides.