Medicinal Plants Camphor Chemical Type Formation Mechanism Of Basic Research

Chemical type belongs to infraspecific variance, which means that the same kind of plant is divided into various types because of their difference chemistry composition. There isn't obvious character of morphology among different kinds of chemical types. It is a biodiversity phenomenon in infraspecific. Chemical type, which is the result of actions of genotype and environment, is the all-pervading phenomena in medical plants. Because of the different efficacy of chemistry types in medical plants, the chemical type can affect the efficacy of medicine plants in clinic. The research of chemical type is very important for insuring the surity and efficacy of medical plants, evaluating the quality of medicine plants and empoldering medical plants. But now we don't know how different chemical types come into being.The Cinnamomum plants are important economic trees which contain many kinds of fragrant oil. Chemical type is the all-pervading phenomena in Cinnamomum plants. The Cinnamomum plants often mixed up in the same area, which contain different chemical types, are unable to be distinguished from the shape. The oils of branchs and leaves of plants mix each other, which make the main content of the necessary composition not high. So we must increase the cost to get the qualified products by the complicated separation. In addition, offsprings of sexual reproduction take place variance in the oils of leaves and branchs. The variance is different because the main content is different. So it is very important to research how different Chemical types of the Cinnamomum plants come into being.We find five kinds of chemical types of Cinnamomum camphora (L).Presl grow in the same area in Ji'an, Jiang Xi Province, P.R.China. The effect of macroscopical ecological environment is identical, but the effect of microcosmi cecological environment maybe different. We found different kinds of endophytic fungi in leaves of different kinds of chemical types in our ago research. Endophytic fungus is the portion of microcosmi cecological environment, which abroad distribut in roots, leaves, stems, fruits, seeds, and so on. Endophytic fungus can stimulate formation and accumulation of the plant sondary metabolite. In addition, we must study the functional genes related to secondary metabolite of plants in inheritance aspect becase the formation of chemical type is dirtectly related to secondary metabolites.We use two kinds of chemical types of Cinnamomum camphora (L). Presl, namely Cinnamomum camphora chvar. Borneol and Cinnamomum camphora chvar. Linalool, as the model plant to study the formation of chemistry type. We study the endophytic fungus in leaves of Cinmmomam camphora chvar. Bomeol and terpene synthase genes in Cinnamomum camphora chvar. Linalool. We aim at establishing foundation on expounding the formation mechanism of five kinds of chemical type of Cinnamomum camphora (L).Presl in the future. The main contents and results are summarized as follows:1．To study the oils chemical composition of leaves of Cinnamomum camphora chvar. Bomeol sexual reproduction offspringsWe collect one hundred seeds from ten trees and make seeds germinate. We transplant seedling in the flowerpot to cultivate after fifteen days. We analyze the oils of growing seedling with GC and GC-MS in different period. The results are as follows:(1) The process of seeds germinateing: seeds first are treated for 3 min with vitriol, then soak this seeds warmly at 25℃until seeds appear cleft and take this seeds into incubator to cultivate at 25℃.(2) After cultivated for three months, the concents of the leaves oils is very simple. Seedlings come into being two kinds of chemical types, the main content of leaves oils of two kings of chemical types is safrole or methyleugenol, but borneol is very little.(3) After cultivated for twelve months, there are two kinds of chemical types among the leaves of seedlings. But the main content of the leaves oils of chemical types is different from the main content of the leaves oils of seedling cultivated for three months. This two kings of chemical types are borneol and 1．8-cineol.2．To study the endophytic fungus in leaves of Cinnamomum camphora chvar. BorneolTwenty five endophytic fungi isolates were separated and purified from leaves of Cinnamomum camphora chvar. Borneol. We analyzed volatility contents of endophytic fungi by GC and GC-MS. We found volatility contents of endophytic fungus ji12 including borneol and indole ramification. We identified the endophytic fungus ji12 by analyzing the ITS region sequence. The endophytic fungus ji12 ITS region has about 610bp, which is high identity to cochiobolus nisikadoi ITS region. We inferred endophytic fungus ji12 belongs cochiobolus .So we deduced that the endophytic fungus is related to the chemistry type formation of Cinnamomum camphora chvar. Borneol.3．To study the encoding genes of terpene synthase in Cinnamomum camphora chvar. LinaloolWe designed primers according to those conservative regions of geraniol synthase genes in Cinnamomum tenuipitum Kosterm and linalool synthase genes in other plants. We used those primers to clone the interest gene fragments using genome DNA as the template. Then we used those primers, whicn can clone fragment from the genome DNA, to amplify the terpene synthase encoding gene fragments using cDNA as the template. Thus, we can validate the interest gene fragment amplified from DNA and cDNA template. According to the amplified fragment, we designed primers to amplify the 3'-end and the 5'-end of the interest gene fragment by RACE technique. The results are as followed:(1) We cloned two terpene synthase DNA fragments from DNA, which denominated CTSR-1 and CTSR-2．The aligned sequences of CTSR-1 and CTSR-2 are about 700bp in length and have high identity to eraniol synthase genes in Cinnamomum tenuipitum Kosterm. At one time, we also amplified two terpene synthase cDNA fragments, which denominated namely CTSR-3 and CTSR-4．The aligned sequences of CTSR-3 and CTSR-4 are about 700bp in length and have high identity to eraniol synthase genes in Cinnamomum tenuipitum Kosterm.(2) Sequences of CTSR-1 and CTSR-2 were aligned using DNAMAN. There is high identity between the exons reigon of CTSR-1 and CTSR-2, But there is low identity between the intervening region of CTSR-1 and CTSR-2．So we can identify different kinds of chemical types on the intervening region of terpene synthase genes.(3) We amplified the 3'-end of CTSR-3 and CTSR-4 by 3'-RACE technique. The aligned sequences of 3'-end of CTSR-3 were about 1．0 kb in length and has 143bp superposition region with CTSR-3．We blast the fragments in GenBank and found it has high identity to eraniol synthase genes in Cinnamomum tenuipitum Kosterm. The aligned sequences of 3'-end of CTSR-4 were about 1．2kb in length and h and has 181bp superposition region with CTSR-3．We blast the fragments in GenBank and found it has high identity to eraniol synthase genes in Cinnamomum tenuipitum Kosterm. we amplified a short gene fragment and GGPP reductase gene fragment. The short gene fragment is a part of 3'-end of CTSR-3．GGPP reductase gene fragment has not superposition region with CTSR-3 or CTSR-4, which is denominated CGGPPR.(4) We amplified two terpene synthase fragments by 5'-RACE method, which are about 440bp or 340bp in length. Those two fragments have 71bp superposition region with CTSR-3．The fragment which is about 440bp in length, CTSR-3 and 3'-end of CTSR-3 are combined and blasted in GenBank. The combine fragment has high identity to eraniol synthase genes in Cinnamomum tenuipitum Kosterm. The fragment which is about 440bp in lenth belongs to CTSR-3, but it don't include initiation codon. The fragment which is about 340bp in length was blast in GenBank. It has high identity to eraniol synthase genes in Cinnamomum tenuipitum Kosterm. We combined it with CTSR-3 and 3'-end of CTSR-3, but the combined fragment can not be readthrough. So the fragment which is about 340bp in length doesn't belong to CTSR-3．It belongs to anther terpene synthase gene.We come to a conclusion as followed:1．Sexual reproduction offsprings of Cinnamomum camphora chvar. Bomeol come being into infraspecific chemical variance. Infraspecific chemical variance is related to development of plants. Cultivated for three months, Seedlings come into being two kinds of chemical types, the main content of leaves oils of two kings of chemical types is safrole or methyleugenol, but borneol is very little. Cultivated for twelve months, there are two kinds of chemical types of seedlings. But the main content of the leaves oils of chemical types is different from the main content of the leaves oils of seedling cultivated for three months. Those two kings of chemical types are borneol and 1．8-cineol.2．The endophytic fungal ji12, which was separated from leaves of Cinnamomum camphora chvar. Borneol, can produce borneol and indole derivatives. It demonstrated the formation of Cinmmomam camphora chvar. Borneol may be relataed to endophytic fungal.3．There are many terpene synthase genes in leaves of Cinnamomum camphora chvar. Linalool. Those terpene synthase genes are very conservative. We amplified two terpene synthase DNA fragments and two terpene synthase cDNA fragments from genome DNA and from cDNA, respectively. Those four terpene synthase genes have high identity. So we can amplify different terpene synthase genes from different kinds of chemical types according to the identity reigon of terpene synthase genes. This can help us elucidate the formation mechanism of different kinds of chemical types from heredity.(4) The intervening region of terpene synthase genes has lower identity than the exons reigon high of terpene synthase. So we can identify different kinds of chemical types before chemical type comes into being on the intervening region of terpene synthase genes.