| Aphids are a class of small insects,which belonging to Hemiptera.They can directly feed on phloem sap using its piercing-sucking mouthparts and transmit various plant viruses to cause losses to crop production.They are one of the major agricultural pests.Pea aphid has many typical characteristics of aphids,such as wing polyphenism,body color polymorphism and parthenogenesis as well as sexual reproduction.The complete genome of pea aphid has been published,numerous transcriptome data have been published,making it an ideal target for aphids on the fields of molecular biology research.As a significant technique to study gene function,RNAi has been widely used in insects,but the current study indicates that RNAi efficiency varies greatly among insects,and RNAi efficiency is relatively low among many major insect groups.Those reason seriously impedes the wide application of RNAi technology.The function of multiple genes has been studied using RNAi in pea aphids.However,RNAi efficiency varied greatly among different research results,and some results even had conflicts.Given the diversity and polymorphism of aphids in natural populations,it is not clear whether changes in RNAi efficiency in aphids are related to their different polymorphisms.To elucidate this question,we first selected yellow and white genes,which are commonly used as phenotypic markers in a variety of insects such as fruit flies,and compared the efficiency differences between different combinations of aphids(green winged/wingless and red winged/wingless)using RNAi technique by injected.It was found that dsRNA of the target genes only induced the decrease of the expression level of the target gene in green wingless aphid,and the expression level of the target genes returned to normal on 48 hours after injection,while the expression of related genes in other polymorphic combinations was not affected.Meanwhile,we also detected the expression of core molecules of Sid-1,Ago-2,R2D2,DcR-1 in the RNAi pathway after injected with the above dsRNA in aphids.Only the expression levels of sid-1and Ago-2 genes were up-regulated,but the expression levels of other genes were not significantly changed.These results suggest that there were differences in RNAi efficiency among different polymorphic aphids,but there was no significant correlation between RNAi efficiency and the combination of wing polyphenism,body color polymorphism as well as only partial gene expression in RNAi pathway molecules was correlated with target gene inhibition.Combining the RNAi efficiency of these two genes in different polymorphisms,we found that the RNAi efficiency was generally lower in all polymorphisms of aphids,suggesting that some nucleases in hemolymph may be involved in the degradation of extracellular dsRNA.Taking green aphid as the object,we further studied the degradation ability hemolymph of different wing polyphenism in vitro for different genes dsRNA.It was found that the degradation velocity of dsRNA was closely related to the content of related enzymes in hemolymph.High concentration of hemolymph extracts could degrade dsRNA rapidly.Compared with winged aphids,the degradation rate of dsRNA was slightly slower in hemolymph of wingless aphids.The above experimental results suggested that low RNAi efficiency in pea aphids was a common phenomenon in different polymorphisms,and related nucleases in hemolymph might be the key to affect RNAi efficiency.Therefore,transcriptome analysis was performed to compare the expression of related genes after dsRNA injected after different times,and combined with the genome-wide screening method of aphid,we attempted to find relevant nucleases that might affect the RNAi efficiency of aphid.However,the transcriptome analysis have not found any related gene of nuclease,but we found 3 potential genes by the method of genome-wide screening,and has been preliminarily identified in the expression of two potential nuclease in hemolymph by RT-PCR and its specific function remains to be further in-depth research.The above two studies showed that although RNAi efficiency was different among different polymorphic aphid,RNAi efficiency was relatively low on the whole,which greatly hindered the research on the gene function in pea aphids using this technology.CRISPR/Cas9 technology based on DNA editing provided us with a new opportunity.At present,CRISPR/Cas9 technology has been successfully applied for gene editing in a variety of insects.Existing experiments have shown that one of the key points for the success of CRISPR/Cas9 in insects is to inject sgRNA and Cas9 protein into eggs before blastocyst stage.However,the populations of pea aphid are generally parthenogenesis in the laboratory,so to gene editing experiment of aphids,the first is inducing sexual aphids.Sexual aphid mating,spawning,and aphids eggs must usually takes about three months after hatching fundatrices,so to clear the spawn rule of pea aphids is one of the key to carry out the CRISPR/Cas9 experiment.The male and sexual female aphids were induced by different low temperature and short light respectively.After sexual aphids mate in the different sex ratio of one female one male and two females one male,the results suggest that from the first egg laying,the number of eggs per day per female in one female one male is all higher than the ratio of two two females one male from first to twelfth day,but the daily number of layingeggs per female was relatively low on the two ratio.Therefore,acquirement of enough male and egg-laying female aphids is the key for collecting sufficient early embryo especially before the blastocyst stage.In addition,the number of laying eggs by one female one male in light was higher than dark,while there was no difference in the number of laying eggs on the other ratio.Then we tested the hatching rate of eggs at different time after low temperature diapause,the results indicated that hatching rates under 4? with 90,100 and 110 days are52.3%,49.2% and 57.6% individually.No significant difference was found among these treatments.Considering the experimental period and the losing of egg during the diapausing period,eggs incubated at 4? for 90 days was selected as the diapausing time in applying CRISPR experiment.To sum up,there were differences in RNAi efficiency among different morphs pea aphid,but generally showed low RNAi efficiency.The nucleases in hemolymph that can rapidly degrade dsRNA may be one of the key factors affecting the efficiency of pea aphid RNAi,rherefore,further research on the function of related enzymes will help to clarify this problem.One of the keys to study aphid gene function using CRISPR/Cas9 technology is to obtain eggs before blastocyst stage.However,the low average daily spawning number of pea aphid suggest that adequate sexual females and males are the key to obtain adequate eggs.This study laid an important foundation for accurately evaluating the application of RNAi technology in aphid gene function research and promoting the application of novel CRISPR/Cas9 technology in aphid functional gene analysis. |
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