| Tomato powdery mildew,as a worldwide fungal disease,the harm to tomato production is increasing year by year.MLO gene family,as an important susceptible factor to powdery mildew,has an important negative regulatory effect on powdery mildew resistance in plants.Previous studies show that SlMLO2 and SlMLO4 genes were closely involved in the stress response of powdery mildew,but the related functions and mechanisms are not clear.Therefore,this study carries out bioinformatics analysis on SIMLO2 and SlMLO4 genes,the susceptible variety Moneymaker(MM)is used as the test material,SlMLO2 and SIMLO4 genes are cloned,the expression of these two genes in 7 tissues including root,stem,leaf,flower,green fruit,color changing fruit and red fruit is analyzed by real-time fluorescence quantitative PCR,and Expression level of SlMLO4 in different period after induced by powdery mildew;constructing of SlMLO2 and SlMLO4 gens overexpression vectors based on pCAMBIA2300-GFP vector,transient transformation of Nicotiana benthamiana for subcellular localization analysis,agrobacterium mediated genetic transformation of Nicotiana benthamiana,susceptible tomato varieties MM and resistant varieties 62579 and 62794;Constructing SlMLO4 gene knock-out vectors based on CRISPR-Cas9 technology,genetic transformation of MM.The main results are as follows:1.Multiple sequence alignment shows that there are six conserved domains in SlMLO2,SlMLO4 and their homologous sequences;Phylogenetic analysis shows that the most homologous sequence with SlMLOs family is Solanaceae plants;The prediction of protein physicochemical properties shows that the molecular weight of SlMLO2 protein is 57459.05Da,the theoretical isoelectric point is 9.01,and contains 6 transmembrane helices;the molecular weight of SlMLO4 protein is 62562.53Da,the theoretical isoelectric point is 8.47,and contains 7 transmembrane helices;Subcellular localization prediction results shows that SlMLO2 and SlMLO4 proteins are located on the cell membrane.2.The cloning vector sequencing results shows that SlMLO2 and SlMLO4 genes are consistent with the submitted sequences of NCBI.SlMLO2 gene has a total length of 1515bp,encoding 504 amino acids;SlMLO4gene has a total length of 1659bp,encoding 552 amino acids.3.Tissue specific studies show that,using the expression level of the color-changing fruit as a control,the expression level of the SlMLO4 gene in stems,flowers,roots,leaves,green fruits and red fruits is about 36 times,28 times,22 times,9 times,2.5 times and 1.5 times that of color-changing fruits;Using the expression level of the stems as a control,the expression level of SlMLO2 gene in red fruit,flower,color-changing fruit,root,leaf and green fruit is about 3 times,3 times,2.9 times,1.8 times,1.4 times and 1.4 times of stems.4.The expression level study of SlMLO4 gene induced by powdery milde shows that,the expression of SlMLO4 gene was up-regulated from Oh to 12h after inoculation of powdery mildew;the expression of SlMLO4 gene at 3h after inoculation of powdery mildew had no significant change compared with Oh,and the expression at 6h and 12h after inoculation was about 3 and 10 times of 0h.5.Plasmid sequencing results proves that the successful construction of SlMLO2,SlMLO4 gene overexpression vector and SlMLO4 gene editing vector.6.By transiently transforming Nicotiana benthamiana,the subcellular localization results show that pCAMBIA2300-SlMLO4 is localized on the cell membrane,and pCAMBIA2300-SlMLO2 is localized on the cell membrane and nuclear membrane.7.Regeneration plants of pCAMBIA2300-SlMLO2,pCAMBIA2300-SlMLO4 and pCAMBIA2300-GFP in Nicotiana benthamiana and tomato(MM,62579,62794)are obtained.The transformation of MM tomato by SlMLO4 gene editing vector is in rooting stage.The transforming efficiency results show that the transforming efficiency of MM and 62559 is significantly higher than that of 62794. |
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