| Achyranthes bidentata Blume belongs to the Achyranthes L.,a perennial deep-rooted dicotyledonous herb.A.bidentata has a variety of medicinal effects,and is used as medicine with dried roots.It is one of the famous "four medicines" in Henan Province.Oleanolic acid triterpene saponins and Ecdysterone are used for A.bidentata the main active ingredient,and its biosynthetic pathway is the Mevalonate Pathway（MVA）,which was regulated by a variety of enzyme genes.For example,squalene synthase（SS）,β-amyrin synthase（β-AS）and cycloartenol synthase（CAS）.In order to increase the content of the main medicinal components in A.bidentata,the function of the AbSS gene in the MVA pathway was studied by molecular biology methods,hoping to increase the content of the main medicinal components in A.bidentata.Our group constructed the AbSS overexpression vector by seamless cloning method.The overexpression vector was transferred into the model plant Arabidopsis and A.bidentata by the Agrobacterium infection method,and AbSS expression level in transgenic Arabidopsis and A.bidentata were determined by the q RT-PCR method,HPLC method to detect changes in oleanolic acid and ecdysterone content in transgenic strains.WE also studied the effect of overexpression of AbCAS gene in A.bidentata callus on the content of oleanoic acid and ecdysterone.The main findings are as follows:1.Using seamless cloning technology,we obtained pART-CAM-SS and pCAM35s-GFP-SS recombinant plasmids successfully.2.The plant subcellular localization vector pCAM35s-GFP-SS was transiently transformed into tobacco epidermal cells.The results showed that A.bidentata SS protein was mainly existed in the cytoplasm and nuclear membrane.3.The overexpression vector pART-CAM-SS was transfected into A.thaliana using the flocculent dip method,screening and PCR verification to obtain the T3 generation homozygous overexpressing the AbSS gene in A.thaliana.Select 2,3,8 and 12 A.thaliana for research.Compared with the control,tthe transgenic Arabidopsis is more robust,and the fresh weight,dry weight and plant height of the AbSS transgenic Arabidopsis were significantly higher.q RT-PCR detection revealed that At SS genes in various tissues were significantly increased,especially in roots,flowers and fruit pods.In addition,the expression levels of related enzyme genes At HMGR1、 At FPS、At SE、Atβ-AS and At CAS in the MVA pathway in transgenic Arabidopsis also significantly increased.Compared with the control,the content of oleanolic acid in transgenic A.thaliana 2,3 and 12 increased by 2.05,1.45,and 2.17 fold,and the content of ecdysterone in 2,3,8 and 12 increased by 1.22,1.34,0.76 and 0.57 fold,respectively.4.The AbSS overexpression vector was transferred into the leaves of A.bidentata,and callus cells were obtained by induction culture.After screening and PCR verification,transgenic positive callus cells were obtained,and SS-1、SS-3、SS-4 and SS-7 were selected.Four AbSS-positive cell lines with faster cell division and better growth were used as materials for continued culture research.The q RT-PCR method was used to detect the gene expression of AbSS,Ab HMGR,Ab FPS and Ab SE,Abβ-AS and AbCAS in the positive callus.Compared with the control,the expression of AbSS and the expression of related genes in the MVA pathway also increased significantly.The content of oleanolic acid and ecdysterone in the callus of the transformed AbSS A.bidentata was determined.The content of oleanolic acid of SS-1、SS-3、SS-4and SS-7 was 1.56、1.81、2.48 and 1.96 fold than the control,the ecdysterone content was 1.79,1.61,1.44 and 1.51 fold than the control,respectively.5.Transferred AbCAS into A.bidentata and obtained 4 positive cell lines with better growth,namely CAS-1、CAS-5、CAS-7 and CAS-9.The q RT-PCR method detects the expression of AbCAS and related upstream genes.Compared with the control,the expression of AbCAS related upstream genes were increased significantly.In the transformed AbCAS callus of A.bidentata,the ecdysterone content of CAS-1、CAS-5、CAS-7 and CAS-9 all increased significantly compared with the control,while the content of oleanolic acid decreased significantly.Among them,the content of ecdysterone in CAS-1 was the highest,reaching 0.46 mg/g,which was 2.47 fold that of the control group,while the content of oleanolic acid in CAS-5 was the lowest,which was a decrease of 118.38% compared with the control group.Based on the above results,the main conclusions of this study are as follows:1.A.bidentata SS protein mainly exists in the cytoplasm and nuclear membrane of tobacco epidermal cells.2.The A.thaliana and A.bidentata callus lines of overexpressing AbSS were obtained,and the expression of AbSS gene in the two transgenic lines was significantly increased.3.The plant height,fresh weight,dry weight and chlorophyll content of transgenic A.thaliana increased significantly,indicating that AbSS has an impact on the growth and development of plants.4.Transformed AbSS A.thaliana and A.bidentata callus cell lines oleanolic acid and ecdysterone significantly increased,indicatied that AbSS played a key role in the biosynthesis of triterpene saponins and phytosterols.5.A callus line with over-expressing AbCAS A.bidentata were obtained,and the expression of AbCAS in the transgenic cells was significantly increased.The content of ecdysterone in the overexpressing AbCAS A.bidentata callus cell line was significantly increased,while the content of oleanolic acid was significantly decreased,proved that the AbCAS gene could regulate the synthesis of phytosterols and can inhibit the flow of 2,3-epoxidized squalene to the triterpene saponin synthesis pathway. |
