Primary Screening Of Disease-resistance Mutants Of Dendrobium Officinale And Preliminary Study On Resistance Mechanism Based On RNA-seq

ObjectiveDendrobium officinale Kimura et Migo originates from Dendrobium?Orchidaceae?and it is traditionally used as a valuable traditional Chinese herbal medicine.It has the functions of nourishing stomach,nourishing yin and clearing away heat.Its main effective components are Dendrobium polysaccharides,alkaloids,phenols,amino acids,etc.Modern medical research shows that D.officinale has anti-tumor,anti-aging,and enhanced immunity,blood sugar lowering and effective reduction of liver damage.The wild D.officinale grows slowly and its natural reproduction ability is extremely low.Due to excessive excavation and continuous destruction of the ecological environment in recent years,resources are on the verge of extinction.Studies have shown that the differences in the contents of polysaccharides and total alkaloids from cultivated D.officinale mainly come from genetic variation,and the lag in species selection is the bottleneck of the current development of D.officinale.The insects and diseases of D.officinale are more serious,and its yield and quality are greatly reduced.The investigation found that anthrax is most common in the D.officinale planting area,with serious damage,and it is highly contagious and difficult to control,seriously affecting the yield and quality of D.officinale.For the prevention and control of anthrax,the current measures are mainly to improve the cultivation environment,drug control and so on.Because there are no varieties that are immune to diseases such as anthrax,chemical control causes problems such as pesticide residues and resistance to pathogens.Coupled with the lag in research on breeding techniques and the lack of new,disease-resistant varieties,the development of D.officinale has been slowly.The adoption of modern breeding techniques to speed up the breeding process of new varieties of D.officinale has been imminent.Therefore,this study used EMS mutation breeding methods combined with RNA-seq technology to study the mechanism of interaction between D.officinale and anthracnose pathogens,in order to lay a foundation for the cultivation of new varieties of D.officinale.MethodsIn this study,EMS was used to mutate the protocorm of D.officinale and the crude toxin of pathogenic bacteria was used to screen D.officinale resistant seedlings.RNA-seq technology was used to study the difference between D.officinale before and after the infection to investigate the infestation of D.officinale and Colletotrichum gloeosporioides interaction mechanism.Specific tasks include:1.EMS mutagenesis of protocorm of D.officinale was performed at different concentrations and for different times.The LD₅₀ value was obtained according to the survival rate,and the appropriate mutagenic concentration was determined by combining with the variation;the ISSR-PCR was used to detect the variation from the molecular level,and the comprehensive morphology was used.Statistical observations were conducted to establish a preliminary EMS mutagenesis mutant library of D.officinale;the pathogen inoculation method was screened,and the pathogen was inoculated into D.officinale EMS mutagenized transplanted seedlings to detect the resistance of EMS mutagenesis mutants to C.gloeosporioides.2.Explore the influence of crude toxin of C.gloeosporioides on the growth of D. officinale.Use D.officinale normal tissue culture seedlings with different concentrations of Colletotrichum gloeosporiorum crude toxin for resistance induction and screening to select the resistance concentration.The EMS-induced mussels of D.officinale were used to screen out resistant mutants of D.officinale that were resistant to the crude toxin of C.gloeosporioides.3.The spore suspension of Colletotrichum gloeosporioides was inoculated on the normal transplanted seedlings of D.officinale by acupuncture inoculation method to obtain three different susceptible stage samples after infestation.The RNA-seq technique was used to explore the infestation of Colletotrichum gloeosporioides.Differences and interaction mechanisms before and after D.officinale.Results1.D.officinale protocorm treated with different EMS mutagenic concentrations and different mutagenesis time,the treatment group with the mortality rate closest to the LD₅₀was found to be EMS concentration 1.0%mutagenesis treatment for 6 hours,which can be used as EMS mutagenizing D.officinale Appropriate mutagenesis treatment;and by trait observation and ISSR-PCR detection,D.officinale mutated by EMS after different mutations and a large genetic similarity coefficient,can obtain a higher mutation rate;through the screening of the pathogen inoculation method,The method of acupuncture inoculation of spore suspension of C.gloeosporioides can be used to infect pathogens of D.officinale.It can be used to detect the disease resistance of D.officinale seedlings induced by EMS;9 groups of mutant seedlings were selected and inoculated with the seedlings of the control group.The spore suspension ofC.Gloeosporioides showed that the mutant plants had more disease resistance than the control plants,indicating that the EMS mutagenesis of D.officinale could be used for breeding and breeding of resistant plants.2.Studies on the effect of crude toxins of C.gloeosporioides on the growth of rotocorm from D.officinale showed that the crude toxin of anthrax has a certain biological activity and virulence,which can replace the pathogens to study the resistance screening of the plants;At 40%?C8?,the mortality of protocorm from D.officinale was the highest and the seedlings appeared chlorosis and withering.Therefore,C8 can be used as a selective pressure for resistance and combined with EMS to induce D.candidum to obtain resistant plants.3.The pathogenic bacteria were inoculated on the leaves of healthy D.officinale by rubbing inoculation method.According to the infection rate and infection degree,the susceptible leaves samples were selected on the 7th,14th and 21st days after infection.The RNA-Seq technology was applied to D.officinale.The healthy leaves and the three different stages after infection were studied to obtain the differential genes and GO enrichment function among different samples,and the interaction mechanism of C.gloeosporioides infestation with D.officinale was preliminarily understood.ConclusionIn this study,the EMS mutagenesis,traits observation and molecular detection methods of D.officinale were used to determine the effectiveness and reliability of EMS mutagenesis for the mutation breeding of D.officinale,and D.officinale resistant plants could be screened from them.At the same time,D.officinale resistant plants were also obtained by applying the crude toxin of C.gloeosporioides to the tissue culture medium of D.officinale and EMS mutagenesis;the samples before and after infection with D.officinale by RNA-Seq technology were used.A differential gene study was conducted to understand the interaction mechanism between C.gloeosporioides and D.officinale.This study integrated the mechanism of EMS mutation breeding and plant-pathogen interaction in D.officinale,in order to provide a certain research basis for disease-resistance breeding of D.officinale and cultivation of new varieties.