Study On The Lateral Root Development And Preliminary Screening Of Regulator Gene Of Radix Bupleuri

Radix Bupleuri roots are commonly used as one of herbs in medicine.Bupleurum rooteffective components is saikosaponins,which have anti-inflammatory,liver,antitumor and other pharmacological activities.Significant difference are found in different varieties of bupleurum root structure.For example,Bupleurum chinense has multi branch,less rootlet and single branc hroot structure.Bupleurum scorzonerifolium has brown root and white rootstructure.Pharmacological experimental study showed that different bupleurum root structure had significant difference on medicinal ingredients.In this study,the lateral root primordia development were observed and the genes related to the root formation were cloned in“Chuanbeichai No.1”and“Chuanhongchai No.1”,and the specificity of the these genes were analyzed between the auxin processing time gradient.Finaly,the related genes to lateral root formation in“Chuanbeichai No.1”and“Chuanhongchai No.1”were screened,which laid the foundation for further determining the key genes that influence the root formation.The results were as follows:1.Analysis on the lateral root primordia formation process of“Chuanbeichai No.1”and“Chuanhongchai No.1”Paraffin sectioning experiment showed that the lateral root primordia formation of“Chuanbeichai No.1”started near the pericycle cells,and part of the endodermis cells also participated in this formation.This process could be divided into five stages:monolayer stage;2³ cells layers stage;multiple cells layers stage;conical lateral root primordia stage;breakthrough epidermal cells stage.“Chuanhongchai No.1”lateral root development was similar to“Chuanbeichai No.1”,except endothelial cells participated.The results showed that Radix Bupleuri lateral root development not only had species conservatism but also species specificity.2.The influence of growth regulating substances on Radix Bupleuri lateral root developmentDifferent concentrations of growth regulating substances had significant difference on Radix Bupleuri lateral root development.This study showed that auxin and calcium ions promoted lateral root development,but auxin inhibitors and calcium ion chelating agent inhibited it.10?mol/L IAA and Ca²⁺greatly increased the lateral root development of“Chuanbeichai No.1”,similarly,0.01?mol/L IAA and 1?mol/L Ca²⁺greatly increased the lateral root development of“Chuanhongchai No.1”.Besides,0.01?mol/L TIBA also greatly promoted the lateral root development of“Chuanbeichai No.1”,but inhibited“Chuanhongchai No.1”.Furthermore,the calcium chelating agents?EDTA?mainly inhibited the root development of Radix Bupleuri.3.Analysis of AUX/IAA,HO and LBD genes's cloning and sequencingAuxin-responsive gene?AUX/IAA?,Heme oxygenase-1 gene?HO?and Lateral organ Boundaries-Domain gene?LBD?were selected as candidate genes for PCR cloning and sequencing.In addition to LBD gene is not cloned successfully for the primers,the rest of the genes were cloned successfully.4.Expression analysis of AUX/IAA?HO and LBD genes during lateral root formationThe above experiments showed that the most optimal concentration of IAA with“Chuanbeichai No.1”was 10?mol/L,and with“Chuanhongchai No.1”was 0.01?mol/L.On the basis of adding auxin,the specificity of the these genes were analyzed between the auxin processing time gradient by real-time fluorescent quantitative PCR.Four genes were selected by SPSS statistics software:IAA27-CH-c9124?IAA11-CH-c3456?HO-CH-c4076 and HO-CH-c5308,which jointly participated in the development process of the root system.5.Bioinformatics analysis of regulatory genesStatistical analysis showed that there were four genes related to lateral root development.AUX/IAA super family included two genes:IAA27-CH-c9124 and IAA11-CH-c3456.Bioinformatics analysis showed that the homology of IAA27-CH-c9124 base sequence is 94%,and the amino acids by IAA27-CH-c9124gene encoded were the alkaline protein.The highest content of protein secondary structure was alpha helix.However,the homology of IAA11-CH-c3456 base sequence is 95%,and the amino acids by IAA11-CH-c3456 gene encoded were the neutral protein,the highest content of protein secondary structure was?-pleated sheet.Sequence analysis showed that the amino acids by IAA27-CH-c9124 and IAA11-CH-c3456 gene encoded belonged to the AUX/IAA super family.The evolutionary tree clustering analysis showed that IAA27-CH-c9124 and IAA11-CH-c3456 gene were mostly affinity with carrot.In addition,there were two genes in HO super family:HO-CH-c5308 and HO-CH-c4076.Bioinformatics analysis showed that the homology of HO-CH-c5308 base sequence is 96.2%,the homology of HO-CH-c4076 base sequence is 96.7%.The amino acids by HO-CH-c5308 and HO-CH-c4076 gene encoded were the alkaline protein,and the highest content of protein secondary structure was alpha helix.Domain analysis showed that the amino acids by HO-CH-c4076 gene encoded belonged to the HO super family,but HO-CH-c5308 was not.The evolutionary tree clustering analysis showed that HO-CH-c5308 gene were mostly affinity with pepper,and there were evolutionary relationship of HO-CH-c4076 in“Chuanbeichai No.1”and“Chuanhongchai No.1”.