Identification And Preliminary Function Analysis Of The Bemisia Tabaci Q (Gennadius) Salivary Calcium-Binding Protein

Bemisia tabaci?Gennadius?is a worldwide agricultural pest,which directly and indirectly damages host plants by sucking mouthparts to feed on the phloem sap of host plants,secreting honeydew,and spreading plant viruses,thus affecting the growth and development of host plants and causing serious economic losses to agricultural production.As a component of the calcium signaling pathway,calcium-binding proteins achieve their biological functions by binding to calcium ions,a ubiquitous and essential second messenger in all eukaryotes,to regulate cell signaling and cell life cycle.Plant pathogen associated molecular patterns in perception,Ca²⁺elevation is one of the earliest signaling events in plants.When the calcium ion concentration is high,the sieve molecules are dispersed and swell,blocking the juice circulation.When the concentration is low,the sieve molecules shrink and the juice is smooth.Calcium-binding protein can maintain the continuous uptake of insects from the phloem screen by reducing the calcium ion concentration.Calcium-binding protein as a candidate effector may play an important role in the interaction between Bemisia tabaci and host plants.In order to understand the role of calcium binding proteins in Bemisia tabaci Q,this paper conducted a preliminary exploration on the functions of calcium binding proteins in bemisia tabaci.Firstly,the gene cloning,sequence analysis,phylogenetic tree construction and cell localization of calcium-binding protein were carried out on the calcium-binding protein of Bemisia tabaci.Then the expression patterns of calcium-binding protein in different tissues,different ages,different hosts and different time periods after starvation induction were analyzed.Finally,the calcium-binding protein was subjected to the T.glutamate-mediated transient expression experiment and the RNAi technology was used to interfere with the calcium-binding protein.Bioassay analysis was performed on the survival rate and fecundity of Bemisia tabaci.The main research results are as follows:1.Bioinformatics analysis and localization of calcium-binding protein?1?The full length of the calcium binding protein gene of Bemisia tabaci was obtained by gene cloning.The full-length sequence of the open reading frame?ORF?was669 bp.Sequence analysis showed that the calcium-binding protein of Bemisia tabaci was a protein of 222 amino acids with a predicted molecular mass of 24.55 kDa and a theoretical isoelectric point pI of 4.64.signal peptide was 21 amino acids and did not have a transmembrane signal.It has an EF-hand chimeric conserved domain with a calcium binding site near the C-terminus of the amino acid sequence.?2?The phylogenetic tree was constructed using the amino acid sequence deduced from the calcium binding protein by Neighbor-joining in the MEGE6.0 software and the amino acid sequence of other insect calcium binding proteins published by NCBI.The results of phylogenetic tree analysis indicated that:The calcium-binding protein of Bemisia tabaci was closely related to the Hemiptera insect,Sipha flava and Melanaphis sacchari,and belonged to the same branch,and Lepidoptera insect Helicoverpa armigera and Vanessa tameamea,Spodoptera litura and the Hymenoptera insect Monomorium pharaonis,Linepithema humile,and Trachymyrmex cornetzi have the longest evolutionary relationship.?3?A calcium-binding protein subcellular localization experiment was performed.Calcium-binding protein was inserted into pCAMBIA1300?GFP?expression vector to form calcium-binding protein::GFP fusion protein,which was transformed into Agrobacterium GV3101 by electric shock and then injected into tobacco.Under the laser scanning confocal microscope,the calcium-binding protein of Bemisia tabaci was observed in N.benthamiana.Subcellular localization in tobacco leaf cells.The results show that the calcium binding protein is localized in the nucleus and cell membrane of tobacco.2.Expression pattern of calcium binding protein?1?The expression of calcium-binding protein in different tissues of Bemisia tabaci was analyzed by real-time PCR.The results showed that calcium-binding protein was expressed in all tissues of Bemisia tabaci,and the expression of calcium-binding protein in the thorax,abdomen,leg and wing of Bemisia tabaci was 0.5985,0.8529,0.6897,and0.5067 times of the expression of calcium-binding protein in the head,respectively.The expression of calcium-binding protein in the head of Bemisia tabaci was the highest,which was significantly higher than that of other tissues of Bemisia tabaci;the expression of calcium-binding protein in the wing was the lowest,significantly lower than that of other tissues of Bemisia tabaci.The expression of calcium-binding protein in other tissues of Bemisia tabaci;the expression level of calcium-binding protein in the abdomen was significantly higher than that in the thorax,leg and wing;There was no significant difference in the expression level of calcium-binding protein in the thorax and leg amount.?2?The expression of calcium-binding protein in different developments of Bemisia tabaci was analyzed by real-time PCR.The results showed that calcium-binding protein was expressed in all stages of growth and development of Bemisia tabaci,and the expression of calcium-binding protein was the lowest in the egg stage of Bemisia tabaci,which was significantly lower than other ages.The expression levels of calcium-binding protein in first and second instar,third instar,fourth instar and adult stage were 4.38,5.47,16.76 and 5.03 times than the expression of calcium-binding protein in the egg,respectively.The highest expression in the fourth instar nymph was higher than that in other stages.There was no significant difference in the expression levels of calcium-binding proteins in the three stages of the first and second,third,adult.?3?The expression of calcium-binding protein in different time periods after starvation-induced feeding was analyzed by real-time PCR.The results showed that the expression of calcium-binding protein in adult adults of Bemisia tabaci was significantly increased after starvation-induced feeding.The expression of the calcium-binding protein was significantly higher in adults of Bemisia tabaci at all time points?1 h,2 h,3 h,4 h,5h,12 h?than that of the adults of Bemisia tabaci,which were fed for 0 h,which were 2.12,1.6291,1.7077,1.9114,1.5438 and 1.7066 times,respectively.The expression of calcium-binding protein in adults of Bemisia tabaci in 1 h and 4 h was significantly higher than that at other time points?0 h,2 h,3 h,5 h,12 h?,but there was no significant difference between the two expression levels.?4?The expression of calcium-binding protein in Bemisia tabaci feeding on different hosts was analyzed by real-time PCR.The results showed that the expression of calcium-binding protein of Bemisia tabaci in pepper and tomato was slightly higher than that of Bemisia tabaci in the cotton,but there was no significant difference among the three hosts.3.Preliminary validation of of the function of calcium-binding protein?1?pEDV::calcium-binding protein expression vector was constructed using the Burkholderia glumae type III secretory system,which was transformed into calcium-binding protein expression vector by electric shock to perform the transient expression reaction of calcium-binding protein in tobacco.As can be seen from the changes in the phenotype of tobacco,the injection of negative control pEDV without load would cause changes in the phenotype of tobacco,resulting in programmed necrosis in tobacco leaves.In addition,the injection site of calcium-binding protein also showed programmed necrosis failed to inhibit the plant defense response caused by pEDV without load.?2?Screening of calcium-binding protein RNAi interference primers.The expression levels of calcium-binding protein in Bemisia tabaci were analyzed by quantitative real-time PCR using four pairs of interfering primers dsCa-F1,dsCa-F2,dsCa-F3 and dsCa-F4 to interfere with Bemisia tabaci at 2 and 3 days.DsCa-F2 of the interference efficiency was the highest and the most stable when interfered for 3 days.dsCa-F2 was selected as the interference primer for interfering with the expression of calcium-binding protein of tobacco powder,and the bioassay was performed after 3 days of interference.?3?The survival rate of Bemisia tabaci after the interference of calcium-binding protein RNAi was measured.The survival rate of Bemisia tabaci was analyzed after 3days of dsEGFP and dsCa interference on the tomato seedlings for 3 days..The results showed that the survival rate of Bemisia tabaci after dsCa interference was slightly lower than that of the control dsEGFP,but there was no significant difference between the two.?4?The fertility of Bemisia tabaci after the interference of calcium-binding protein RNAi was measured.After 3 days of dsEGFP and dsCa interference Bemisia tabaci was placed on tomato seedlings for 3 days.The results showed that the single female oviposition of Bemisia tabaci after dsCa interference was significantly lower than that of the control dsEGFP,and the single female average fecundity of Bemisia tabaci after dsCa interference was 9.35,while the single female average egg production of Bemisia tabaci after dsCa-EGFP was 12.88.