| The Watermelon mosaic virus is one member of potyviruses, transmitted by aphids with nonpersistent type,extensively spreaded over the world, and mainly cause mosaic diseases of watermelon and melon. In resent years,the disease severely took place, which had become the one of formost factors that limited high and stable yields of watermelon and melon. In this paper, the complete nucleotide sequence of watermelon mosaic virus Chinese Isolate was determined. On the basis, the HC-Pro protein and CP protein both are the factors for aphid transmission were cloned and expressed in vitro, and their antiserum were prepared. By far western blot, viral receptors were identified in aphids, and their interactions were studied. The molecular mechanism of nonpersistent transmission was further elucidated. The results are as follows:1. By RT-PCR and 5′-and 3′-RACE mehtods,the nucleotides of coding region and untranlating region of genome of WMV were cloned. The sequences assemblied, the full-lenth cDNA were acquired. Based on analysis, we find the RNA of WMV is single and positive strand. Excluding 3′-terminal poly(A) tails, the genome is 10037 nts long,including the 5′-UTR and 3′-UTR.A large open reading frame encoded a polyprotein of 3217 amino acids with a predicted molecular weight of 365.8 kD. The initiation codon ATG locates at 133-135 nts, the stop codon UAA locates at 9784-9786 nts.Based on analysis of computer, the structure and correlated and conservative motif of genome of WMV were determinated. The 5′-UTR is 132 nts long, containing more A and less G( The percentage of A is about 46.3%, but G is only about 6.1%), and the 3′-UTR is 254 nts long, containing more T and less C ( The percentage of T is about 40%, but C is only about 13%). The polyprotein can be proteolytized by proteinase to ten mature proteins, which are P1,HC-Pro,P3,6K1,CI,6K2,NIa-VPg,NIa-Pro,NIb and CP from 5′-terminal to 3′- terminal.The nucleotide number of other regions of the genome of WMV-CHN is same as WMV-Fr except the 3′-UTR is two nts more than that of WMV-Fr. Compared with WMV-Fr, the identity of entire genome of WMV-CHN is 92.5%; the polyprotein is 92.4%; the nucleotide and amino acid homologies of different matural prpteins are 87.5%-96.8% and 90.1%-100%, respectively; the NIa-VPg varies most. Aligned with WMV CP of other countries the nucleotide and amino acid homologies of WMV-CHN CP are 93.0%-95.0% and 96.5%-98.6%, respectively. Compared with other potyviruses, there is the highest identity between WMV-CHN with Soybean mosaic virus, the nucleotide and amino acid identity is 78.4% and 86.1%, respectively; the second are Peanut stripw virus, Bean common mosaic necrosis virus, Bean common mosaic viurs, Blackeye cowpea mosaic viurs and Zucchini yellow mosaic virus, the homologies of nucleotide and amino acid are 68.3% and 70.9%, 67.2% and 71.2%, 67.3% and 70.3%, 67.7% and 70.9%, 62.2% and 62.9% in turn, respectively.The 5′-termini of WMV is intimately correlated with evolution of viruses. Based on analysis, the N termini of 5′-UTR of WMV-CHN was found that has a conservative region of about 50bp nts with related SMV, BCMV and PStV those have same nts in 5′-UTR. The N terminal amino acids have higher identity with that of two BCMV and three PStV, and the C terminal amino acids have higher identity with that of SMV, so WMV may is from interspecific recombination of between BCMV or PStV with SMV.2. WMV CP gene was inserted into pET30a digested by EcoRI/Sal I, the expression vector of pET30-WCP was constructed, and transformed into E. coli BL21. Induced by IPTG for 2-8h, the HC-Pro protein with molecular weight of about 37 kD was successively expressed. After induced by IPTG for different time, the CP protein began to express after 4h, and the yields were the largest after 8h. The purified protein induced as antigen was used to immunize the rabbit, and the antiserum of WMV CP was prepared whose titer measured by ELISA was 1:6400; western blot analysis indicated that the antiserum could serologically react with CP.3. WMV HC-Pro gene was inserted into pET30a digested by EcoRI/Sal I, the expression vector of pET30-WHC was constructed, and transformed into E. coli BL21. Induced by IPTG for 2-8h, the HC-Pro protein with molecular weight of about 57 kD was successively expressed. After induced by IPTG for different time, the HC-Pro protein began to express after 2h, and the yields didn't increase after 8h. The purified protein induced as antigen was used to immunize the rabbit, and the antiserum of WMV HC-Pro was prepared whose titer measured by ELISA was 1:6400; western blot analysis indicated that the antiserum could serologically react with HC-Pro.4. WMV HC-Pro gene was inserted into pPIC9K digested by EcoRI/NotI, the eukaryotic expression vector of pPIC9K-WHC was constructed; the recombinant was digested by Sal I and transformed into Pichia pastoris GS115, the high copy transformants with Mut+/His+ phenotype were obtained. Induced by 1% methanol for 5d, the HC-Pro secrete protein with molecular weight of about 66 kD was successively expressed. Western blot analysis indicated that the secrete protein could serologically react with antiserum prepared by HC-Pro expressed in E. coli.5. By far western blot method, six obvious and repeated proteins that can bind bait protein HC-Pro were identified in total proteins of Mynus persicae can transmitting WMV, the molecular weight was 86kD, 84kD, 66kD, 63kD, 48kD and 46kD, respectively, of which 66kD and 63kD proteins relative strongly reacted; Only one relatively obscure protein was identified in total proteins of Psammotettix apicalis not transmitting WMV; But CP used as bait protein, no protein was identified in total proteins of both insects.6. The interactions of HC-Pro and CP were studied by far western blot method, the results indicated that CP and HC-Pro expressed in E. coli could specially bind on NC film, which not only verified the functions that CP binding HC-Pro could help aphids transmission, but first reported the factors for aphids transmission expressed in vitro had the same biological functions as natural proteins isolated from plants. When the interactions of receptors, HC-Pro and CP were studied, HC-Pro couldn't bind CP any longer after it bound receptors.Summarizing above all, in this paper, the genome of WMV was analized, and HC-Pro and CP used as bait protein, several proteins related aphids transmission were screened in aphids, which provide test foundations for the molecular mechanism of nonpersistent transmission.
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