Comparative Analysis Of Transcriptome Of Two Glycyrrhiza Species In Response To Salt Stress And Establishment Of Their Shoot Apex-based Genetic Transformation System

Soil salinization is one of the main stresses restricting agriculture and animal husbandry production in China.Wild plant species growing in salinized environments have evolved a series of unique and complex mechanisms of stress resistance during the long-term evolutionary process.Glycyrrhiza inflata and G.uralensis are typical perennial herbs of Glycyrrhiza in Leguminosae,whose original habitats are salinized soil.Both of these two Glycyrrhiza species have strong salt tolerance.G.inflata is the most salt-tolerant Glycyrrhiza species,exploring the physiological mechanisms and relevant molecular basis of its adaptation to salt stress could lay an important theoretical foundation for the genetic improvement of forages and crops.It was found that G.inflata and G.uralensis can resist oxidative damage under salt stress by enhancing the enzymatic reactive oxygen species(ROS)scavenging system,and the ability for eliminating ROS in G.inflata is stronger than that in G.uralensis.Meanwhile,G.uralensis has a relatively higher growth rate than G.uralensis under salt stress.Hormones such as auxin,cytokinin and gibberellin play an important role in promoting plant growth.It had been suggested that plants could form various adaptive mechanisms,including ROS scavenging system and hormone regulation,etc.,via the responses at the transcriptome level under salt stress.Therefore,comparing the response patterns of different species to salt stress at the transcriptome level is of great significance to further elucidate the mechanisms of stress resistance in plants with relatively stronger salt tolerance and to investigate their stress-resistant gene resources.However,the comparative studies on enzymatic ROS scavenging system and hormone regulation between G.inflata and G.uralensis at the transcriptome level have not been reported.In addition,it is necessary to establish an efficient and stable genetic transformation system of G.inflata,in order to further analyze the function and mechanism of genes involved in salt tolerance of G.inflata via molecular biology techniques,such as RNA interference and overexpression.However,there is still no report on the establishment of genetic transformation system for G.inflata.In this thesis,we compared and analyzed the transcriptome of G.inflata and G.uralensis in response to salt stress,in terms of ROS scavenging and the regulation of hormones,focusing on three important hormones(auxin,cytokinin and gibberellin)with the function of promoting plant growth,and then preliminarily screened out the gene resources in G.uralensis that may be related to its salt tolerance.Meanwhile,a shoot apex-based genetic transformation system for G.inflata and G.uralensis was established.The main results are as follows:1.Compared with that in G.uralensis,the expression of nine genes encoding Trx in the root as well as one gene encoding NRX,two genes encoding Trx,two genes encoding GST and three genes encoding GRX in the leaf of G.inflata were up-regulated under 200 m M Na Cl,which may be one of the reasons for its stronger ability of ROS scavenging under salt stress.2.The decrease amplitude in auxin content in the leaf of G.inflata under salt stress was less than that of G.uralensis.Compared with that in G.uralensis,the expression of AO and YUCCA10 involved in auxin synthesis increased in the leaf of G.inflata.The expression of ARF2,ARF4 and ARF18,the transcription repressors in auxin signaling pathway,was significantly reduced,while the expression of genes encoding four repressors of ARF,AUX22,IAA14 and two AUX28,were up-regulated in the root of G.inflata under salt stress,which may contribute to enhancing auxin signal transduction in the root.3.The decrease amplitude in cytokinin content was less than that in G.uralensis,and the expression of LOG1,LOG7,LOG8 and CYP735A1 involved in cytokinin synthesis was up-regulated in the root of G.inflata under salt stress.Meanwhile,the expression of ARR5 and ARR9 in the root of G.inflata were reduced compared to that of G.uralensis,which probably help to promote the signal transduction of cytokinin in the root of G.inflata under salt stress.There was no significant difference in the expression of genes associated with cytokinin metabolism and signal transduction in the leaf of the two Glycyrrhiza species under salt stress.4.Under salt stress,the changes of gibberellin content in the root or leaf of G.inflata and G.uralensis were the same.Compared with G.inflata,the genes encoding RHT-1 and GAI1 involved in gibberellin signal transduction were down-regulated in the root while two homologous genes encoding GID1 B gibberellin receptor were induced in leaf of G.uralensis.This might be conducive to enhancing gibberellin signal transduction in G.uralensis to adapt to salt stress.5.Using 4-day-old G.inflata and G.uralensis seedlings as explants,the shoot apex-based genetic transformation system mediated by Agrobacterium was established,and the positive transgenic lines with GUS reporter gene were successfully obtained through this method.The optimum content of quartz sand which could wound stem apical meristem and significantly affect the infection efficiency during genetic transformation in this method was 140 g/L.The transformation efficiency of G.inflata and G.uralensis with this content of quartz sand was 2.41% and 6.85%,respectively.