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Cloning And Expression Characteristics Analysis Of The Thaumatin-like Protein Gene From Bursaphelenchus Xylophilus(Bx-TLP1) And Pinus Massoniana(Pm-TLP)

Posted on:2016-01-26Degree:MasterType:Thesis
Country:ChinaCandidate:J WangFull Text:PDF
GTID:2393330482475528Subject:Forest Protection
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Pine wilt disease(PWN),one of the world's four major forest diseases,is able to make the susceptible plants withered quickly perish,and the spread of this forest disease is very fast.Since entering China,it threats to the production of Forestry and does harm to biodiversity,severe forest ecological benefits,resulting in huge economic losses.With the whole genome sequence of PWN determined in 2011,numerous studies have been quick to carry out in the molecular aspects of this disease.As a pathogenesis-related protein,Thaumatin-like proteins play an importent role in the process of plant defense.However,some researchers found that there are two proteins secreted by pine wood nematode is same to the host pine,one of them is thaumatin-like protein,but the study of their specific funcitions has not been carried out.In this paper,we complete clone of the thaumatin-like proteins from pine wood nematode TLP-1 and the host pine,and finish the analysis of the two gene sequences.At the same time,we detected the expression patterns of the two proteins within the interaction system between Bursaphelenchus xylophilus and Pinus massoniana by real-time PCR technology,the main results are as follows:wo got the full-length cDNA of two proteinsby RT-PCR technique,named Bx-TLPand Pm-TLP.the GenBank accession numbers is KM063438,KM063439.Phyre2 software successfully constructed three-dimensional structural model of the two proteins,and the Reasonable assessment of them detected by Molprobity found that there were 98%,99%of the amino acids located in the very reasonable area.The basic properties of the two proteins are as follows:The cDNA length of thaumatin-like protein from Bursaphelenchus xylophilus is 441 bp,encoding 146 amino acid sequence and 15.9KD protein.isoelectric point pI 7.58,including N-terminal signal peptide structure,is a hydrophilic secreted protein.Second Structure contains 67.8%random coil,10.3%a-helix and 21.9%? fold.This sequence is very similar to the Published sequence from Bursaphelenchus xylophilus Genome Database,the GenBank accession numberis BUX.s00036.89,the similarity is up to 98.87%,and there are five base differences with them.Except the Bases on the 330 bit changed amino acid from threonine to alanine,others located on 26,29,44,100 bit sequence,which Encoded amino acids were same to the BUX.s00036.89.But compared to other species,this protein is more similar to the western white pine Pinus monticola TLP protein(ADB97933.1),the E-value of 2e-04 with the blastp in NCBI.Howrer,the E value of Bx-TLP with the THN-3 of Caenorhabditis elegans was higher than 10(the smaller of the E-value number,the similar with two sequence.The Pm-TLP protein's cDNA length is 543bp,encoding 180 amino acid sequence and 18.66KD amino acid sequence protein.the GenBank accession numbers is KM063439.The encoded amino acid sequence 18.66KD protein.The isoelectric point pI is 4.40,the second structure containing N-terminal signal peptide.This protein is a hydrophilic secreted protein.Second structure contains 62.22%of the random coil,30.56%of the ?-helix and 7.22%p fold.Phylogenetic analysis revealed that the protein is most similar(91%)to the reproted TLP sequences from western white pine(ADB97931.1,AAS85755.1),and the homology with other TLP sequence of western white pine(ADB97933.1,ADB97932.1)is up to 80%.Inoculationing pine wood nematode suspension on 5 years old Pinus massoniana,in the latter month,we detected the moisture and pinene content determination every 3 days in the latter month.At the same time,we also centrifuged the nematodes from trees and Interception the roots,stems and leaves from the tree to extracted RNA,and do the quantitative fluorescence detection.This study found that,after the pine wood nematode inoculation pine,The moisture content presented up-down-up-down trend,it begans to decline after 15d,and reached the lowest value in 27d.Pinene content and two types of thaumatin-like protein expression patterns all showed up-down-up-down trend,but the time of each other is different.The two highest point of pinene is 9d and 27d;The first highest point of thaumatin-like protein fron pine nematode is 6d,the last piont is 27d;the highest point of the whole class of Pinus massoniana is 9d and 27d,and there is a significant increase in 3d,6d slightly decreased but still higher than control,and the expression of root is more significant than the stem and laeves during the test.We can speculate that the two proteins have mutual restraint function of each other in the interation process by two types of protein expression patterns,and the thaumatin-like protein from host may play an active role in the pinene synthesis pathway,which is based on the function of activate defense pathway of this protein and the dynamic change trend of Pm-TLP and the content of pinene.
Keywords/Search Tags:Bursaphelenchus xylophilus, Pinus massoniana, thaumatin-like protein, gene cloning, expression analysis
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