Full-length Cloning And Expression Analysis Of ACS And ACO Genes, Key Enzymes In The Ethylene Biosynthesis Pathway Of Lonicera Japonic

Lonicera macranthoides is one of the plants belongs to Lonicera genus of Caprifoliaceae family,its expanded buds and early blossoms are the main sources of the traditional Chinese medicine Lonicerae Flos.But because of the short florescence and the mix flowering periods of the conventional type Lonicera macranthoides Hand.-Mazz.,a large number of L.macranthoides resources were wasted because they were difficult to harvest;compared with the conventional variety,the Lonicera macranthoides `Xianglei’ cultival had many good traits such as long bud stage,oderly bud and closed corolla.In order to explore the root cause to the emergence of the excellent characteristics of `Xianglei’ variety,project team ever draw conclusions that the dynamic change trend of ethylene production rate were different in these two varieties of L.macranthoides through experiments,and put forward hypothesis that endogenous ethylene difference might be the cause to the different phenotypes.On the basis of previous studies,this study intends to conduct researches on related genes of key enzymes in ethylene biosynthesis pathway of L.macranthoides Hand.-Mazz.and L.macranthoides `Xianglei’ at the molecular level,so as to find out the functional genes that lead to different phenotypes of these two L.macranthoides.【Objective】To clone the key enzymes genes of ethylene biosynthesis pathway such as ACS and ACO respectively from L.macranthoides Hand.-Mazz.and L.macranthoides`Xianglei’,conduct expression analysis on them,and to provide the research basis for exploring the functional genes which lead the different phenotypes of two varieties of L.macranthoides.【Methods】1.According to the flowering phase classification criteria of L.macranthoides Hand.-Mazz.and L.macranthoides `Xianglei’ founded by project team earlier,identifying and setting the description for each flowering phases with the development degree and appearance shape characteristics of the flower specimen as the indexes.2.Extracting total RNA from the flower,stem and leaf samples of L.macranthoides Hand.-Mazz.and L.macranthoides `Xianglei’ respectively by polysaccharide polyphenols kit method;with the L.macranthoides transcriptome database that the project team obtained before,screening Unigene that is annotated as ACC synthase(1-aminocyclopropane-1-carboxylic acid synthase)and ACC oxidase(1-aminocyclopropane-1-carboxylic acid oxidase)in it;using RT-PCR and RACE technology to clone the full-length cDNA of relevant Unigenes,name and upload them to NCBI;bioinformatics tools are used to analyze and identify the physicochemical property,conserved domain and gene homology of the obtained proteins;finally,using q RT-PCR technique detecting the expression patterns of each target gene in different species of L.macranthoides.【Results】1.The identification and description of each flowering phase of different L.macranthoides were conducted.The conventional strain belongs to the bud-opening type,the characters difference and color changes are so obvious between each flowering period that we can easily distinguish them;the cultivar `Xianglei’ belongs to the bud-closing type,whose flower bud is always closed from the bud appear to fade,the colors changes are unconspicuous between the djacent flowering phases so that are easily to be confused.2.The total RNA of flowers,stems and leaves were successfully extracted from L.macranthoides Hand.-Mazz.and L.macranthoides `Xianglei’,and the RNA purity,concentration and integrity were examined to meet the requirements of subsequent experiments.The full length sequence of ACS3,ACO1,ACO4 and ACO5 cDNA were successfully cloned from these two kinds of L.macranthoides;the names and the Gen Bank accession numbers were as follows,L.macranthoides Hand.-Mazz.:Lm-ACS3(Gen Bank: MH724196),Lm-ACO1(Gen Bank: MK630682),Lm-ACO4(Gen Bank: MH673877)and Lm-ACO5(Gen Bank: MK542553);L.macranthoides`Xianglei’: Lm-XL-ACS3(Gen Bank: MH724197),Lm-XL-ACO1(Gen Bank:MK630683),Lm-XL-ACO4(Gen Bank: MH681654)and Lm-XL-ACO5(Gen Bank:MK542552).3.By conducting bioinformatics analysis on ACS3,ACO1,ACO4 and ACO5 of the two varieties of L.macranthoides,it is found out that the gene sequence and physicochemical properties of the protein between orthologous genes of two varieties have high similarity,but little difference.Between two orthologous genes obtained in this study,the open reading frame(ORF)length are consistent,the ORF length of ACS3,ACO1,ACO4 and ACO5 gene are 1452 bp,1092 bp,1089 bp and 927 bp respectively,and the number of coded amino acids are the same;homologous genes share the same conserved domain,two ACS3 both containing the conserved Aminotran＿1＿2 domain,while ACO1,ACO4 and ACO5 all containing the conserved DIOX＿N and 2OG-Fe II＿Oxy domain;through BLAST and multi-sequence alignment,it find that the amino acids of these two L.macranthoides encoded by ACS3 have high similarities with ACC synthase of other plants in NCBI,while amino acids encoded by ACO1,ACO4 and ACO5 have high similarities with ACC oxidase;and the phylogenetic tree analysis results of cloned genes showed that L.macranthoides Hand.-Mazz.has the most closest affinity with L.macranthoides `Xianglei’;for ACS3,ACO1 and ACO4,most other closely plants were jueged to be dicotyledonous and valvular plants,while ACO5 could only be judged that the plants with close affinity were dicotyledons,but difficult to fit into a specific subclass.4.The q RT-PCR results showed that the expression patterns of ACS3,ACO1,ACO4 and ACO5 genes were different in two varieties of L.macranthoides.In conventional variety,the expression quantity of these cloned genes were changed significantly with the flowering periods,and the overall expression trend were increased,and they all have obvious jump peaks,after reaching the peak,it will decline rapidly at the later stage;while in `Xianglei’ cultival,the expression difference and changes between the adjacent flowering stages is relatively small.【Conclusion】1.There were significant differences in flower phenotype and the length of flowering duration between L.macranthoides Hand.-Mazz.and L.macranthoides`Xianglei’,and it could classify and identify their variety and the flower stage according to the characteristics.2.Relevant genes in transcriptome database could be successfully cloned by RT-PCR and RACE technology,according to the results of bioinformatics analysis,the cloned genes were ACS and ACO genes in the biosynthesis pathway of L.macranthoides.3.Combined with bioinformatics and expression analysis results,it was found that the gene sequence and protein physicochemical properties between two orthologous genes cloned in the study have high similarity,but there were different expression patterns.Among them,ACS3,ACO1 and ACO4 genes might be main functional genes involved in regulating the ethylene biosynthesis of L.macranthoides.ACO5 may be involved in the regulation of ethylene production too,but may not the main cause for soaring ethylene production in the aging period.4.This study provided theoretical basis for the isolation and identification more endogenous ethylene-related genes from L.macranthoides,and provided experimental basis for further verifying the biological functions of these genes.