| With the rapid development of facility agriculture in our country,severely restricts the persistent use of horticulture and need to be solved urgently.To adopt the reasonable rotation,interplanting,intercropping is effective way to solve the obstacles of continuous cropping in vegetables.The results from our research group showed that,as a companion crop,potato onion could suppress Fusarium wilt of tomato and enhance tomato growth performance,increase production 5~8%.There are many reports about the study of the potato onion allelopathy of molecular mechanism,genes regulate potato onion allelopathy,the mechanism is still unclear.In this paper,potato onion and tomato were taken as experimental materials,used high-throughput sequencing technology,digital expression spectrum results that our research group has been identified,obtained allelopathy related genes Chi and Pal gene.Full length of the Acad Chi、Acad Pal was cloned from potato onion using 3’RACE method.We constructed the over-expression vector for Chi and Pal to perform transgenic experiment into Arabidopsis thaliana.We validated the Chi and Pal gene function,the phenotypic changes,Arabidopsis thaliana rhizosphere microorganism community and diversity with PCR-DGGE,q PCR and Illumian Miseq sequencing methods,validated Arabidopsis thaliana root exudates impacting on allelopathic properties of tomato and antagonistic properties.A research platform was constructed for gene cloning and functional analysis in potato onion,in this study to provide a theoretical basis for the allelopathic related genes,it can also provide ideas and technical basis in allelopathy of potato onion.Main results of this paper were as flowing:1.Validated genes expression of potato onion roots of different cultivar according to the detection by real time quantitative PCR,Chi and Pal with the variation of the sampling time,the relative expression amount increased and then decreased after reduction,gene expression and field experiment are basically the same trend.Based on these results,Chi and Pal were selected for the cloning and transgenic study.2.A novel full length Chi and Pal genes were isoleted from patato onion using RCR-RACE and RT-PCR method.The c DNA of Acad Chi has an open reading frame of 633 nucleotides,which encodes 210 amino acids.The c DNA of Acad Pal has an open reading frame of 2124 nucleotides,which encodes 708 amino acids.We constructed the overexpression vector p CAMBIA1301-Chi(Pal)for Chi and Pal to perform transgenic experiment.3.Transgenic Arabidopsis thaliana of Chi gene were flowering time earlier,is to promote the transition from vegetative to reproductive growth.Moreover,impacted changes of leaf;Transgenic Arabidopsis thaliana of Pal gene were flowering time earlier,impacted changes of leaf colors.4.The determination of the final product of the flavonoid biosynthetic pathway showed that the flavonoids content of leafs and roots in the Ac Chi+,Ac Pal+ transgenic Arabidopsis thaliana was significantly higher than that the wild type plants(p<0.05);The determination of the leaf chlorophyll contents showed that,the chlorophyll contents of leafs in the Ac Pal+ transgenic Arabidopsis thaliana was significantly higher than that wild type plants and Ac Pal-mutant(p<0.05).Examined the expression patterns of Chi,Pal genes in different tissues,the expression in the Ac Chi+,Ac Pal+ transgenic Arabidopsis thaliana was higher than that the wild type plants.Their expression in leaves was higher than the other two tissues,followed by stems,and the expression in roots was the lowest.5.DGGE and q PCR results showed that,Transgenic Arabidopsis thaliana of Chi and Pal gene affected Arabidopsis thaliana rhizosphere microorganism community structure and community abundances The Shannon diversity index and evenness index for the bacterial communities,were calculated,and both were significantly higher in the rhizospheres collected from the Ac Chi+ transgenic and wild type Arabidopsis thaliana than the ones collected from the Chi-mutant Arabidopsis thaliana(p<0.05);however,the evenness index for the fungal community was calculated and was found to be lower in the rhizospheres from the Ac Chi+ transgenic Arabidopsis thaliana than in the Chi-mutant or wild type Arabidopsis thaliana(p<0.05).The bacterial community abundances in the rhizospheres was increased significantly in the Ac Chi+ transgenic and wild type Arabidopsis thaliana compared to the Chi-mutant Arabidopsis thaliana(p<0.05);however,the fungi community abundances was decreased in the rhizosphere from the Ac Chi+ transgenic Arabidopsis thaliana compared to those from the Chi-mutant A Arabidopsis thaliana and wild type Arabidopsis thaliana(p<0.05);The bacterial community abundances in the rhizospheres was increased significantly in the Ac Pal+ transgenic Arabidopsis thaliana compared to the Pal-mutant Arabidopsis thaliana(p<0.05).6.Results of Illumian Miseq sequencing show that,Transgenic Arabidopsis thaliana of Chi and Pal gene affected Arabidopsis thaliana rhizosphere microorganism diversity,their community structures were distinguished from each other completely,changed richness index,diversitly index and composition in soil bacterial community.Transformation of Chi gene increased the abundance of Cyanobacteria,Gemmatimonadetes,Gammaproteobacteria,Cyanobacteria;Transformation of Pal gene increased the abundance of Proteobacteria,Alphaproteobacteria,Deltaproteobacteria and Anaerolineae.The principal axis(PCo A)and principal components(PCA)analysis showed that the soil bacterial community structure was changed by different Arabidopsis thaliana rhizosphere.For bacterial community structure,among with mutant Arabidopsis thaliana and wild type Arabidopsis thaliana were distance of transgenic Arabidopsis thaliana.These suggested that were larger differences in bacterial.Transformation of gene affected Arabidopsis thaliana rhizosphere microorganism community structure.The LEfSe analysis received bacterial community of significant differences.That types in dominant flora of bacteria of Ac Chi+ transgenic Arabidopsis thaliana soil,mainly in Cyanobacteria,Xanthomonadaceae,Xanthomonadales,Rhodobacteraceae,Rhodobacterales.That types in dominant flora of bacteria of Ac Pal+ transgenic Arabidopsis thaliana soil,mainly in Alphaproteobacteria,Cyanobacteria,Chloroflexi,Myxococcales,Deltaproteobacteria,Anaerolineales,Anaerolineae.7.Arabidopsis thaliana root exudates of different cultivar stimulated growth of tomato seedlings,Ac Chi+ transgenic Arabidopsis thaliana and Ac Pal+ transgenic Arabidopsis thaliana root exudates significantly stimulated the growth of tomato radicle length,fresh length and dry weight.Arabidopsis thaliana root exudates significantly inhibited the mycelia growth of tomato vertucullium wilt.The colony diameter of tomato vertucullium wilt was decreased significantly in the Ac Chi+ transgenic Arabidopsis thaliana,Chi-mutant Arabidopsis thaliana and wild type Arabidopsis thaliana compared to CK;the colony diameter of tomato vertucullium wilt was decreased significantly in the Ac Pal+ transgenic Arabidopsis thaliana compared to other treatment.The results showed that,we validated the function of Chi and Pal genes from Potato onion.After the transferring,it was found that the morphology of Arabidopsis thaliana was changed first.Meanwhile,the content of flavonoids was increased in the Ac Chi+ and Ac Pal+ transgenic Arabidopsis thaliana.Transformation of Chi and Pal gene affected Arabidopsis thaliana rhizosphere microorganism community structure and community richness,transformation of Chi and Pal gene affected Arabidopsis thaliana rhizosphere microorganism diversity,their community structures were distinguished from each other completely.The Ac Chi+ transgenic Arabidopsis thaliana and the Ac Pal+ transgenic Arabidopsis thaliana had the allelopathic effects and inhibitory effects.Then,were improved allelopathic effects and inhibitory effects of Arabidopsis thaliana. |
PDF Full Text Request