Response Of The Branching Characteristics To Hormones In Red Clover(Trifolium Pratense L.)and Analysis On The Inhibition Function Of Gene TpMAX1

Red clover(Trifolium pratense L.)is one of the most important forages in leguminous plants,which is the strategic material for promoting the national dairy industry revitalization plan and the basic raw material for the development of grass and animal husbandry.Breeding high-yield and high-quality red clover varieties has become one of the main measures to promote the high-quality development of grass and animal husbandry.Branching characteristic is an important agronomic trait of red clover,which determines biomass and plant type,while phytohormones play a key role in regulating plant branching characteristics.Therefore,researching on phytohormones and branching genes that regulate the branching characteristic of red clover,screening and identification of key branching candidate genes,and applying them to red clover breeding research by means of molecular measures will be of great significance to broaden the breeding resources of red clover,improve the plant type and biomass of red clover,and to cultivate new varieties of red clover with high yield and stable yield.Therefore,in this study,two kinds of red clover with more branches(MB)and fewer branches(FB)were used as experimental materials to determine the phenotypic traits and endogenous hormone content.The expression pattern of branch-related genes and branching phenotype results under the regulation of exogenous hormones was analyzed,and the gene TpMAX1,a kind of gene that encodes cytochrome P450 monooxygenase was screened,cloned and genetically transformed into Arabidopsis thaliana for functional verification.In addition,the regeneration ability of different red clover materials was compared,and the best transgenic recipient material of red clover was obtained,and the influencing factors of Agrobacterium-mediated genetic transformation were studied,which provided the foundation for functional analysis of the TpMAX1 gene and transgenic breeding of red clover.The main results are as follows:1 Compared with FB,the number of branches and hay yield of MB plants were significantly increased,and the plant height was significantly decreased.The average ABA,GA3,5-DS,and GA3/ZT of MB plants were significantly higher than that of FB at the early flowering stage.Among different tissues,the average ZT content in axillary buds,the average IAA content in stems,the ABA,5-DS,IAA/ZT,and ABA/ZT in leaves,and the GA3 and GA3/ZT in roots were the highest.Correlation analysis showed that branch number had an extremely significant positive correlation with ABA,GA3,ABA/ZT,and GA3/ZT,and a significant positive correlation with IAA,5-DS and IAA/ZT.2 Exogenous IAA significantly promoted the number of branches of MB,but significantly inhibited the plant height.The synthetic SL analogues rac-GR24 and ABA significantly inhibited the number of branches and plant height of MB,and GA3 significantly inhibited the number of branches of MB,but significantly promoted the plant height.Real-time quantitative PCR was used to analyze the differential transcription responses of branching-related genes under different exogenous hormone treatments.The results showed that exogenous IAA,rac-GR24,ABA,and GA3 could induce the expression of TpMAX1,TpD27,TpCCD8,TpD14,TpDELLA,TpSPS,and TpRAX3 in red clover.In addition,the relative expression level of TpMAX1 in axillary buds was significantly changed,and the expression pattern of this gene was opposite to the branching phenotype under the corresponding exogenous hormone treatments.This indicates that TpMAX1 is not only closely related to the branch development of red clover but also more sensitive to exogenous hormones.3 Compared with FB,the expression level of the gene TpMAX1 was significantly up-regulated in all tissues of MB,with the highest expression level in leaves,followed by axillary buds and stems,and lowest in roots.A red clover gene TpMAX1 encoding 541 amino acids and 1623 bp in length was cloned by PCR.Bioinformatics analysis showed that the relative molecular weight of TpMAX1 protein was 61.38 k D,the theoretical isoelectric point(PI)was 8.62,and the instability index was 38.11,which belonged to a stable protein.Subcellular localization predicts that TpMAX1 may be localized at the endoplasmic reticulum(ER).The secondary structure was mainly composed of α-helix(47.13%)and random coil(37.34%),which was highly consistent with the predicted results of its spatial structure.Phylogenetic tree analysis showed that red clover TpMAX1 protein belongs to the same clade as Medicago truncatula Mt MAX1 and Cicer arietinum Ca MAX1,and they are most closely related to each other.The recombinant expression vector PCAMBIA2300-TpMAX1 was transferred into wild-type Arabidopsis thaliana by the Agrobacterium-mediated floral-dip method,and 8 transgenic Arabidopsis thaliana plants were obtained.Compared with the wild-type Arabidopsis thaliana,the branch number of the transgenic Arabidopsis thaliana was significantly lower than that of wild-type.4 The hypocotyls of red clover line(R),Trifolium pretense cv.Minshan(M)and‘Ganhong No.1’(G)were used as explants to analyze the effects of different genotypes,media,and hormone ratios on the induction and differentiation of healing tissues,and elongation of adventitious shoots and root production,and the results showed that at the callus induction stage,the callus rate of M was the highest(94.67%),and the optimal induction medium was MS+2 mg/L 2,4-D+0.6 mg/L 6-BA.At the callus differentiation stage,the germination rate of G was higher(8.00%),and the optimal medium for differentiation was MS+3 mg/L 6-BA+0.3mg/L NAA.At the rooting stage,the rooting rate of R was higher(92.33%),and the optimal rooting medium formulation was 1/2MS+2 mg/L IBA.Combining the callus rate,bud differentiation rate,and rooting rate of the three red clover materials,it was concluded that‘Ganhong No.1’ had the highest regeneration ability.In view of this,red clover ‘Ganhong No.1’ was used as the recipient material for genetic transformation,and the influencing factors of Agrobacterium-mediated genetic transformation were studied.The results showed that the yellowish dense red clover calluses were inoculated with Agrobacterium solution containing PCAMBIA2300-TpMAX1 plasmid(OD600=0.7)for 20 min,and cocultured for 3 d in dark,and then transferred to differentiation medium containing 300 mg/L Cef to successfully induce resistant buds.On this basis,the Agrobacterium-mediated genetic transformation system of red clover needs further research and exploration.