AM真菌调控黄花铁线莲生长和有效成分含量的机制研究

发布时间：2018-07-23 20:02

【摘要】：丛枝菌根真菌,即AM真菌(Arbuscular Mycorrhiza Fungi,AMF),是一类广泛分布于土壤生态系统的有益微生物,可与90%的高等植物形成AM共生体,菌根。AM真菌能促进植物对营养元素和水分吸收,改善植物的营养状况,从而促进植物的生长发育,提高植物次生代谢产物的积累和抗逆性。黄花铁线莲(Clematis intricata Bunge)为毛茛科铁线莲属草本植物。在中药、蒙药、藏药中作为一种重要的药用植物而被利用,其主要活性成分为黄酮类,有抗炎、镇痛和抗肿瘤活性,且毒性很小。本课题采用湿筛倾析法,研究内蒙古7个地区黄花铁线莲根际土壤AM真菌多样性;在盆栽条件下研究了不同氮(N)、磷(P)水平下接种摩西球囊霉(Glomus mosseae)对黄花铁线莲生长、生理生化特性和总黄酮积累以及利用qRT-PCR技术分析黄酮类生物合成途径关键酶基因响应不同N、P水平,探讨AM真菌对黄花铁线莲生长和有效成分含量积累的影响机制;利用RACE(Rapid Amplification of cDNA Ends)技术克隆黄花铁线莲黄酮类生物合成途径的两个关键酶基因,苯丙氨酸解氨酶基因(CiPAL)和查尔酮合成酶基因(CiCHS),并对其进行生物信息学分析。本课题为规模化种植中蒙药材提供了菌根学和分子生物学依据。主要研究结果如下:1.从内蒙古7个地区的黄花铁线莲根际土壤中,共分离出3属29种AM真菌,其中球囊霉属Glomus 27种,盾巨孢囊霉属Scutellospore 1种,无梗囊霉属Acaulospora 1种。结果表明,黄花铁线莲根际土壤中AM真菌多样性丰富。2.盆栽条件下,不同N水平下接种AM真菌对黄花铁线莲生长有显著影响。不同N水平下,AM真菌均能侵染黄花铁线莲根系,并且能够有效促进黄花铁线莲的生长,提高产量;适N条件下,AM真菌通过影响过氧化物酶活性和渗透调节物质脯氨酸含量,促进黄花铁线莲生长;通过调控黄酮生物合成途径的相关酶基因的表达(其中CiCHS的表达量显著上调)而促进地上部分总黄酮的积累,能够有效改善黄花铁线莲的药材品质。高N则限制了黄花铁线莲生长,降低有效成分含量。因此,施N量为10%N~20%N时,接种AM真菌效果最好。3.盆栽条件下,不同P水平下接种AM真菌对黄花铁线莲生长有显著影响。不同P水平下AM真菌均能侵染黄花铁线莲根系,并且能够有效促进黄花铁线莲生长,提高黄花铁线莲产量;适P水平下,接种AM真菌通过影响过氧化物酶活性和渗透调节物质脯氨酸含量,降低叶片丙二醛含量,使黄花铁线莲更好地生长;通过调控黄花铁线莲黄酮生物合成途径关键酶基因的表达(其中CiPAL和CiCHS的表达量显著上调),促进地上部总黄酮积累,改善了黄花铁线莲药用品质。综合考虑,施P量为5%P~20%P时,接种AM真菌效果最好。4.利用RACE技术,克隆得到CiPAL和CiCHS两个基因的cDNA全长序列。其中,CiPAL cDNA序列全长为2386 bp,含有1个2118 bp的完整ORF,编码705个氨基酸。在CiPAL的第187-202位,含有一个酶活性中心序列(GTITASGDLVPLSYIA),是典型的PAL家族成员;CiCHS cDNA序列全长为1445 bp,含有1个1191bp的完整ORF,编码396个氨基酸。CiCHS含有CHS-like和CHS酶家族两个保守功能域,是典型的CHS家族成员。5.本研究发现,在适量的N、P水平下,AM真菌能够有效促进黄花铁线莲生长,诱导黄酮类生物合成关键酶基因的表达,提高黄花铁线莲有效成分含量,改善其药用品质。
[Abstract]:Arbuscular mycorrhizal fungi, or AM fungi (Arbuscular Mycorrhiza Fungi, AMF), is a kind of beneficial microorganism widely distributed in soil ecosystem. It can form AM symbiosis with 90% higher plants. Mycorrhizal.AM fungi can promote the absorption of nutrient elements and water, improve the nutritional status of plants, and thus promote the growth and development of plants and improve plant growth. The accumulation and resistance of the product secondary metabolites. Clematis intricata Bunge is a herbaceous plant of the Ranunculus Clematis. It is used as an important medicinal plant in Chinese herbal medicine, Mongolian medicine and Tibetan medicine. The main active ingredient is flavonoids, which have anti-inflammatory, antitumor and antitumor activity, and the toxicity is very small. Wet sieves are used in this subject. The diversity of AM fungi in rhizosphere soil of Clematis from 7 areas of Inner Mongolia was studied by dipping method. Under the pot culture, the growth, physiological and biochemical characteristics and the accumulation of total flavonoids and the key to the analysis of flavonoids biosynthesis by qRT-PCR technology were studied under different nitrogen (N) and phosphorus (P) levels inoculated with Mose balloon mildew (Glomus mosseae). In response to different N and P levels, the enzyme gene was used to explore the effect mechanism of AM fungi on the growth and the accumulation of effective components of Clematis latitum; two key enzyme genes, phenylalanine ammonia lyase gene (CiPAL) and chalcone synthase gene (Ci) were cloned by RACE (Rapid Amplification of cDNA Ends) technology. CHS) and bioinformatics analysis. This subject provides mycorrhizal and molecular biological basis for large-scale cultivation of Mongolian medicinal materials. The main results are as follows: 1. from the rhizosphere soil of the 7 regions of Inner Mongolia, 3 genera and 29 species of AM fungi were isolated, including 27 species of saccule genus Glomus, and Scutellospore of the genus megaspora The 1 species, 1 species of Acaulospora, the results showed that the diversity of AM fungi in the rhizosphere soil of the rhizosphere of the yellow flower Clematis was rich.2. potted conditions, the inoculation of AM fungi under different N levels had a significant effect on the growth of Clematis rhizosphere. Under the different N levels, the AM fungi could infect the rhizosphere of the Yellow clematis, and could effectively promote the birth of the Clematis. Long, increase yield; under N conditions, AM fungi can promote the growth of Clematis by affecting the activity of peroxidase and the content of proline in the osmotic substance, and the accumulation of total flavonoids in the upper part of the ground can be effectively improved by regulating the expression of the related enzyme genes of the flavonoid biosynthesis pathway (and the expression of CiCHS is up significantly up). The high N limits the growth of Clematis and reduced the content of effective components. Therefore, when the dosage of N is 10%N~20%N, the best effect of inoculation of AM fungi on the.3. pot culture, the inoculation of AM fungi under the different P levels has a significant effect on the growth of Clematis Clematis. And it can promote the growth of Clematis Clematis and increase the yield of Clematis. Under the level of P, the inoculation of AM fungi by affecting the peroxidase activity and the content of proline in the osmotic substance, reducing the content of malondialdehyde in leaves, and making the plant of Clematis yellow flower better, by regulating the key enzyme gene of the flavonoid biosynthesis pathway of Clematis. The expression of the expression of CiPAL and CiCHS increased significantly, which promoted the accumulation of total flavonoids in the upper part of the ground and improved the medicinal quality of Clematis Clematis. When the dosage of P was 5%P~20%P, the best effect of inoculation of AM fungi was.4. using RACE technology, and the total length of CiPAL and CiCHS two genes was cloned. The total length of CiPAL cDNA sequence was 2386. P, a complete ORF containing 1 2118 BP, encodes 705 amino acids. In the 187-202 position of CiPAL, it contains an enzyme active center sequence (GTITASGDLVPLSYIA), a typical member of the PAL family; the CiCHS cDNA sequence is 1445 BP, contains 1 1191bp intact ORF, and 396 amino acids contain two conserved functional domains, including 396 amino acids. It is a typical CHS family member.5. this study found that at a moderate amount of N and P, AM fungi can effectively promote the growth of Clematis, induce the expression of the key enzyme genes of flavonoids biosynthesis, improve the content of the effective component of Clematis, and improve its medicinal quality.
【学位授予单位】：内蒙古医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.239

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