Study On The Mechanism Of Growth And The Content Of Active Components Of Clematis Intricata Bunge Regulated By AM Fungi

AM fungi(Arbuscular Mycorrhiza Fungi,AMF)is a kind of beneficial microorganism distributing in the soil ecosystem widely;it can form AM symbiosis with 90% higher plants that is called mycorrhiza.AM fungi can promote the absorption of nutrients and water,improve the nutritional status of plants,thereby promoting the growth and development of plants,improving the accumulation of secondary metabolites and stress resistanceof plants.Clematis intricata Bunge is a herbs of the genus Ranunculaceae.It can be used as an important medicinal plant in traditional Chinese medicine,Mongolian medicine,and Tibetan medicine.The main active ingredient of Clematis intricata is flavonoids which have anti-inflammatory,analgesic and anti-tumor activity and low toxicity.To study the AM fungi diversity in rhizosphere soil of Clematis intricata in 7 regions of Inner Mongolia by wet sieve decantation;To analyse the effects of Glomus mosseae on the growth,physiological and biochemical characteristics,and the key enzyme genes of flavonoid biosynthesis pathway in response to different levels of N and P and toinvestigate the effect mechanism of AM fungi on the growth and accumulation of effective components ofClematis intricata.To identify two key enzyme genes in the flavonoid biosynthesis pathway including phenylalanineammonia-lyase gene(CiPAL)and chalcone synthase gene(CiCHS)by RACE(Rapid Amplification of cDNA Ends)technique,and analyse their bioinformatics.This study provides a scientific basis of mycorrhizal and molecular biology for large-scale cultivation of Chinese and Mongolian herb.The main results are as follows: 1.29 species of 3 genera of AM fungi were isolated from the rhizosphere soil of Clematis intricata in 7 areas of Inner Mongolia.27 species of them belong to Glomus,1 specie belongs to Scutellospore,and 1 specie belongs to Acaulospora.The results showed that the diversity of AM fungi in rhizosphere soil of Clematis intricata was rich.2.In the potted condition,AM fungi had a significant effect on the growth of Clematis intricata in different nitrogen levels.Under different nitrogen levels,the arbuscular mycorrhizal fungi could infect the roots of Clematis intricata,thus promoting its growth and raising the production of Clematis intricata.Under the suitable nitrogen levels,AM fungi affected the peroxidase activities and proline content of Osmoregulation Substances,which madeit grow better;Regulating the expression of genes related enzymes involved in the flavonoid biosynthesis pathway,of which the expression of CiCHS was up-regulated significantly,that promotedthe accumulation of total flavonoids,and improved the medicinal quality of Clematis intricata.The high N level limits the growth and reduces the content of active componentsof Clematis intricata.Therefore,when the amount of N was 10%N to 20%N,inoculation with AM fungi had the best effect.3.In the potted condition,AM fungi had a significant effect on the growth of Clematis intricata in different phosphorus levels.Under different phosphorus levels,the arbuscular mycorrhizal fungi could infect the roots of Clematis intricata,thus promoting its growth and raising the production of Clematis intricata.In the appropriate phosphorus level,AM fungi affected the peroxidase activities and proline content of Osmoregulation Substances and decreased the content of malondialdehyde,which made it grow better;Regulating the expression of genes related enzymes involved in the flavonoid biosynthesis pathway,of which the expression of CiPAL and CiCHS were up-regulated significantly,that promoted the accumulation of total flavonoids,and improved the medicinal quality of Clematis intricata.Taking best effect of AM fungi into consideration,the application of P is 5%P to 20%P.4.Two genes of cDNA full-length sequences of CiPAL and CiCHS were cloned by RACE technology.The full length of CiPAL cDNA was 2386 bp and contained a 2118 bp complete Open Reading Frame(ORF)encoding 705 amino acids.CiPAL was a typical member of PAL family which contained an enzyme active center sequence(GTITASGDLVPLSYIA)at the 187-202 position;The full length of CiCHS cDNA was 1445 bp and contained a 1191 bp complete ORF encoding 396 amino acids.CiCHS was a typical member of CHS family which contains two conserved functional domains of the CHS-like and the CHS enzyme families.5.This study showed that AM fungi could effectively promote the growth,induce the expression of genes key enzymes involved in flavonoid biosynthesis pathway,increase the content of active ingredients of Clematis intricata,and improve its medicinal quality with appropriate amount of N,P fertilizer.