| Monochasma savatieri Franch.ex Maxim is a perennial semi-parasitic herb of the Monochasma in Scrophulariaceae.And distributed in southern China that has been found in JiangXi,JiangSu,and FuJian provinces,on the islands of Jiuzhou and Tiancao,and in Japan and Korea.The whole plant is used to treat the common cold,cough,pneumonia,blood flow problems,toothache and so on.The soil matrix of wild M.savatieri is mostly gravel and stone,and it can be widely parasitic on herbs and woody plants,such as Gardenia jasminoides Ellis,Digitaria sanguinalis（L.）Scop and Loropetalum chinense（R.Br.）Oliver.Previous studies on this plant have focused on its chemical constituents and pharmacological effects.However,the wild resources of the plant suffer from many abiotic and biotic stresses,such as excessive illumination,which leads to the limited distribution of the community.Artificial uncontrolled picking and wild animal activities also lead to the risk of plant extinction.Artificial cultivation is the most effective way to solve this problem.In the past two years,cuttings,wild tending,potted cultivation and other methods have achieved good results for this species.However,soil moisture control during the seedling stage is an unsolved problem,and too much water causes the M.savatieri seedlings to grow very slowly.Furthermore,the role of soil microorganisms in plant growth cannot be ignored.Therefore,the research on soil moisture and soil microorganism is of great significance to the success of artificial cultivation of M.savatieri and the continuous supply of Chinese herbal medicine.In this study,soil moisture content suitable for the growth of M.savatieri was selected by setting different soil moisture content and inoculation treatment of arbuscular mycorrhiza,and the beneficial symbiotic relationship between Rhizophagus irregularis and M.savatieri was preliminarily understood.At the same time,in order to study the molecular biology of M.savatieri,we screened and identified the appropriate internal reference genes,and analyzed the expression of water channel protein gene of M.savatieri,cloned a water channel protein gene and carried out the relevant bioinformatics analysis,which provided the basis for revealing the water transport mechanism between M.savatieri and its host.The main results and conclusions are as follows:（1）The results obtained clearly demonstrate that Rhizophagus irregularis improve plant growth by positively affecting the osmotic adjustment,antioxidant enzyme activity and soil enzymes.Furthermore,semiparasitic plants are highly dependent on their host plant and rely on them for normal life activities.In this study,Rhizophagus irregularis and drought treatment to enhance the root length and lateral roots of seedlings may have significantly contributed to the absorption of nutrients and water and greater drought tolerance through parasitic contact with the host,which is the most important life process for semiparasitic plants.The study demonstrates that the use of Rhizophagus irregularis and appropriate drought treatments have great application prospects for the growth and reproduction of semiparasitic plants M.savatieri under artificial cultivation conditions and will be of great help in maintaining the sustainable utilization of this plant.（2）Six housekeeping genes were selected from the transcriptome database of M.savatieri as candidate internal reference genes.The expression of six genes under different soil water content and arbuscular mycorrhizal conditions were analyzed by three internal reference analysis softwares:geNorm,NormFinder and BestKeeper.From the analysis results,the results of the three analysis methods tend to be consistent:TUB and GAPDH analysis results are close and the expression is the most stable,while ACT expression is the most unstable.The expression analysis of AP-2,CYP and UBQ by three kinds of internal parameter analysis software is slightly different,which may be caused by the different principles of data processing of three kinds of analysis software.In this study,TUB and GAPDH genes were screened under different soil water content and arbuscular mycorrhizal conditions,which could provide reference for the further study of gene function.（3）The results showed that with the increase of soil drought degree,the expression of AQPs in the roots of M.savatieri seedlings was significantly increased,and arbuscular mycorrhiza could significantly reduce the expression of AQPs in the roots of the seedlings,but the difference was that MsTIP2;1 is different from other（4）water channel protein genes,which may be due to the parasitic relationship between M.savatieri and the host plant,the M.savatieri needs to absorb more water and nutrition from the host body to regulate its growth and development,which leads to the increase of MsTIP2;1 expression in the root system,so MsTIP2;1 Clone and bioinformatics analysis were carried out.The 747-bp cDNA of encodes a 41-aa signal peptide at the N-terminus and a mature protein of 207 aa.After elimination of the signal peptide,the molecular weight of the TIP2;1 protein is predicted to be 20863.27Da.The total number of negatively charged residues（Asp+Glu）is 8,with 5 positively charged residues（Arg+Lys）.The aliphatic index is 114.54 and the grand average of hydropathicity（GRAVY）0.869.Protscale results indicated that greatest hydrophilicity and hydrophobicity of the TIP2;1 protein at valine 28 and proline 202,respectively.Amino acid sequence analysis showed that the protein encoded by TIP2;1 gene has a typical SGGHVNPAVTFG sequence in major intrinsic family,and contains a very conservative NPA（Asn-Pro-Ala）domain,which is directly involved in the formation of water channels.Furthermore,transmembrane analysis revealed 5 transmembrane domains.This phylogenic tree clearly clusters MsTIP2;1 polypeptides with the TIPs family.The most homologous proteins are AtTIP2;1,the tonoplast intrinsic proteins isolated from Arabidopsis thaliana. |
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