Establishment Of RPA Detection System For Hlb And Construction Of Expression Vector Based On Citrus Leaf Blotch Virus

Citrus Huanglongbing(HLB)is a devastating disease,which has been seriously threatening the Citrus industry.The pathogen can’t be isolated and purely cultured.Therefor,no effective control agent is available so far.The main prevention and control measures of HLB include timely removal of diseased trees,planting of HLB-free nursery trees and concentrated large area joint defense and control of vectors.Therefore,a rapid and simply used detection method of HLB is in demand.In this study,a rapid detection system of RPA(Recombinase polymerase amplification)for HLB was established.Studies have shown that antimicrobial peptide is helpful to control HLB.Meanwhile,the introduction of cb(Cecropin B)、hbd-4(Homo sapiens defensin beta 4A)、aiia(Autoinducer inactivation)into citrus could enhance the resistance of citrus plants to bacterial diseases.In order to avoid the long period of transgenic breading,we first constructed the expression vector p CLBV202 based on CLBV(Citrus leaf blotch virus),then a few improved constructors p CLBV202-CB/HBD-4/Aii A have been achieved,which could fast and systematic expressed CB/HBD-4/Aii A in lemon.Some attempts have been made to prevent and control HLB by using these vectors.The main research results are as follows:1.Establish RPA rapid detection system for HLBIn order to simplify the detection of HLB,primers were designed based on the conservative nrd B,a five-copy gene in Candidatus.Liberibacter spp..A rapid detection system of RPA(Recombinase polymerase amplification)for HLB was then established by primer screening and reacting condition optimizing.Results:1)specificity:the RPA system can detect HLB with specificity,while other 7 kinds of citrus disease pathogens like Xcc(Xanthomonas citri subsp.citri),have no reaction;2)sensitivity:the RPA sensitivity is 100 times of that of ordinary PCR,which is equivalent to that of real-time fluorescence quantitative PCR.;3)the whole detection process is completed within 25min,which is relatively short;4)a total of 87 suspected samples from Sichuan,Yunnan,Jiangxi and Guangxi provinces were detected,and 31 samples were positive in RPA or real-time PCR detection.The positive detection rate of RPA was 35.63%,which was slightly higher than the 31.03%of normal PCR.The RPA detection system established in this study is simple in operation,short in time,strong in specificity,high in sensitivity,and doesn’t need special equipment,so it is suitable for rapid detection of HLB.2.Construction of expression vector based on CLBVOn the basis of the infection clone CLBV201,subgenomic promoter sequences is inserted after the cp(Coat protein)terminator,and the Sma I restriction site is add,a virus expression vector p CLBV202 was then constructed.To verify the expression function of p CLBV202,gfp(Green fluorescent protein)was inserted into the Sma I restriction site,so that a recombinant expression vector p CLBV202-GFP was constructed.Tobacco and Eureka lemon was inoculated by p CLBV202-GFP,using agrobacterium-mediated innoculation,and systemic green fluorescence in tobacco was observed under ultraviolet lamp,while green fluorescence of lemon leaves was observed under confocal microscope.These results indicated that the expression vector p CLBV202 based on CLBV was successfully constructed,which could be used to express GFP rapidly and systematically in tobacco and Eureka lemon.3.Construction of p CLBV202-CB/HBD-4/Aii A expression vectorThe recombinant expression vector p CLBV202-CB/HBD-4/Aii A was further constructed besad on p CLBV202.The tobacco and Eureka lemon was inoculated by p CLBV202-CB/HBD-4/Aii A using agrobacterium-mediated innoculation,while p CLBV202 was used as control.At 18 dpi,tobacco were inoculated by bacterial wilt,with 3 plants as a group,3times repeated.Results:1)the incidence of tobacco in the experimental group was14.3%,and the disease index was 14,while the incidence in the control group was100%,the disease index was 63,indicating that the incidence and disease index of the experimental group were significantly lower than that of the control group;2)The resistance index of tobacco in experimental group was-2.66,compared with that in control group,resistance was evaluated as HR.Therefor,expression CB in tobacco by p CLBV202-CB can enhance tobacco resistance to bacterial wilt.At 365 dpi,leaves of lemon plants containing p CLBV202-CB and p CLBV202were collected and inoculated in vitro with Xcc,3 times repeat.Results:1)the incidence in the experimental group was 43.5%,which was lower than 67.6%in the control group;2)the number of bacteria in the experimental group 5~1,was significantly lower than that in the control group 5~4;3)the disease index(DI)of the experimental group was 15,and disease resistance(R).In the control group,DI was 28,medium resistance(MR).Therefor,expression CB in lemon plants by p CLBV202-CB can enhance lemons resistance to Xcc.Lemon plants containing p CLBV202-CB and p CLBV202 were tested by Diaphorina citri.for HLB.The results showed that the infection rate of the experimental group was 0,while the control group was 3.45%.p CLBV202-CB was used to express CB in lemon plants,which had a certain control effect on HLB.However,due to the novel coronavirus outbreak,Number of Diaphorina citri.and the transmission time is shorter,the experiment needs to be further scaled up and repeated.In summary,the results show that the expression of CB in tobacco and Eureka lemon plants by using virus expression vector p CLBV202,can improve the resistance of the plants to bacterial diseases.