| Melon?Cucumis melo L.?is an important economic crop in Xinjiang.The main melon variety"Queen"has been popuLar with consumers because of its excellent agronomic traits,but its susceptibility to disease has led to a decline in quality,resuLting in a large area of reduced production.Therefore,improving disease resistance is a problem that urgently needs to be solved in melon production.CRISPR/Cas9 is an acquired immune system,which is present in all archaea and most bacteria.It has been widely used in genetic engineering and genetic breeding of animals and plants.Because CRISPR/Cas9 genome editing technology has the advantages of high efficiency,convenience and low cost,it has attracted much attention.In this study,CRISPR/Cas9 genome editing technology was used to knock out the viral disease susceptible gene eIF4E and powdery mildew susceptible gene MLO in Xinjiang's main muskmelon variety"Queen",in order to obtain broad-spectrum disease-resistant melon variety.The main resuLts are as follows:1.RT-PCR semi-quantitative analysis of muskmelon leaves inoculated with virus and powdery mildew,respectively.The uninoculated plants served as negative controls.The resuLts showed that eIF4E and MLO were inocuLated with virus disease and powdery mildew.The gene expression level was higher than the control.It was presumed that the virus and powdery mildew pathogens needed the assistance of eIF4E and MLO genes to complete the growth and reproduction in melon in the early stage of infection.2.The full-length eukaryotic eIF4E gene is 2807 bp.It contains 5 exons and 4introns.The target rate in the target sequence is 0.5906.The total length of MLO gene in melon is 4500 bp.It contains 15 exons and 14 introns.the target rate in the target sequence is 0.7729.3.The sgRNA box was amplified using the dicotyledon knockout vector pP1C.4as a template,and the recombinant vectors pP1C.4-eIF4E and pP1C.4-MLO were successfuLly constructed.4.Explore the effects of different concentrations of IAA and 6-BA on"Queen"explants.The resuLts showed that when the 6-BA concentration in MS solid medium was 1 mg·L-1,the concentration of IAA was 0.05 mg·L-1,the growth state of the callus was good,so the hormone combination at this concentration had a high induction rate for adventitious buds.5.This experiment determined the"Queen"selection pressure of hygromycin in the medium and the optimal inhibitory concentration of cephalosporins.When the hygromycin was added at a concentration of 10 mg·L-1,the growth of explants was inhibited,10 mg·L–11 could be selected as the critical concentration of antibiotics;when the concentration of cephalosporins at 500 mg·L-1,the inhibitory effect on Agrobacterium was best and did not affect the growth of the explants,500 mg·L-11 can be used as the optimal inhibitory concentration of cephalosporins.6.The recombinant vectors pP1C.4-eIF4E and pP1C.4-MLO were successfully transferred into Agrobacterium GV3101 by freeze-thawing method.After one year of genetic transformation experiments,11 transformed plants were obtained through hygromycin screening.9 strains of positive transformed seedlings were initially obtained from the hygromycin-resistance gene.Sequencing the target sequence of transformed seedlings,The resuLts showed that no gene mutations occurred near the target site.This article provided a new idea for the research on the resistance of muskmelon,which laid a foundation for the further study of the function of muskmelon eIF4E and MLO genes. |
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