| Neoseiulus barkeri(Hughes)(Acari: Phytoseiidae),is an important natural enemy of small plant fluid feeding arthropods in agriculture.It has wide application prospect and important application value to prevent pests and injurious mites.In this thesis,the resistances of N.barkeri after adding amino acids of leucine and histidine to starvation,low temperature and high temperature were studied.The functional of N.barkeri prey on T.putrescentiae after adding amino acids was also tested.Furthermore,high-throughput sequencing technology was applied to analyze the transcriptome information of N.barkeri of adding amino acids and non-adding amino acids.Besides,three heat shock protein genes(Hsps)of N.barkeri were cloned and their induced expression patterns were studied.The results will be helpful to understand the molecular mechanisms of Hsps on the resistance of Neoseiulus barkeri to temperature stress.The main test results are as follows:1.Amino acid nutrient addition can prolong the tolerable time for starvatio and improve the resistance of N.barkeri to high temperature,and increase feeding amounts of N.Barkeri on Tyrophagus putrescentiae.In the absence of food condition at 25?,the longest resistance time to hunger is N.Barkeri(Leu group),the N.Barkeri(His group)is second,the control group with no amino acid was the shortest.When the survival rate of the group was 50 %,it shows that adding amino acid can extend the time of the resistance to hunger.When treated at 40 for 10 h,compared with control group?,the survival rate of leucine rearing N.Barkeri and histidine rearing N.Barkeri were increased by 56.00% and 26.00%,respectively.The result indicated that the amino acid could improve the high temperature resistance of N.Barkeri.Adding the nutrition of amino acids made a difference on the predatory ability and maximum daily feeding amount of N.barkeri to T.putrescentiae.Compared with the control group that was reared without amino acid addition,the predation ability of N.barkeri of leucine and histidine rearing was increased by 17.08% and 31.82%,respectively.The N.barkeri which were fed by adding these amino acids,the curve of function response of N.barkeri to T.putrescentiae all fell in Hulling?,which is the typical invertebrates convex type curve.2.This study analyzed and established the transcriptome sequencing of N.barkeri(CK group,non-adding amino acids)and N.barkeri(Leu group,adding amino acids of leucine)using the high throughput sequencing platform,and assembled the sequencing reads into Unigene using the denovo strategy,and conducted annotation of GO functional classification and KEGG pathway analysis to the Unigene.In N.barkeri(CK group),we obtained 52817486 pairs raw data reads(about 7.92 G)and there were 23893 genes and 86422 transcripts.The 23893 Unigene were annotated using the blast tools,and 7260 of which were annotated to Swiss-Prot,13386 to NR,7567 to Pfam,6078 to KEGG,7420 to KOG and6604 to GO.We obtained 64516490 pairs raw data reads of N.barkeri(Leu group)(about 9.68 G)and there were 34480 genes and 57021 transcripts.The 34480 Unigene were annotated using the blast tools,and 10509 of which were annotated to Swiss-Prot,18556 to NR,11230 to Pfam,8499 to KEGG,10735 to KOG and9664 to GO.Compared with CK group and Leu group of N.barkeri,there were 1291 significant differently expression genes.Among them,the expression of 376 genes were up-regulated,and 915 genes were down-regulated.In this study,the transcriptional study of N.barkeri was carried out,and a large number of gene data were obtained,which provided support for the further study of the environmental conditions on N.barkeri from the molecular level.3.A total of 21 Hsps unigene genes were obtained from the database of Neoseiulus barkeri,including Hsp90,Hsp70,Hsp60,Hsp40 and small Hsp family.There were 10 Hsp90 families,7 Hsp70 families,Hsp60 family has two,Hsp40 family and small Hsp family have one each.The ORF of NbHsp90 contained 2,142 bp,comprising 713 amino acids with calculated masses of about 81.9 k Da and a theoretical isoelectric point(p I)of 5.08.The ORF of NbHsp60 contained 1755 bp,comprising 583 amino acids with calculated masses of about 62.2 k Da and a theoretical isoelectric point(p I)of 5.55.The ORF of NbHsp40 contained 1206 bp,comprising 401 amino acids with calculated masses of about 44.7 k Da and a theoretical isoelectric point(p I)of 6.21.Similarity analysis of the known Hsps amino acid sequences in other species showed a high degree of similarity to the amino acid sequences corresponding to the Metaseiulus occidentalis and other insects.4.Real-time PCR technique was used to examine the expression of NbHsps treated with heat and cold shock.Results showed that the expression of NbHsp90,NbHsp60,NbHsp40 of N.barkeri can be up-regulated by both cold shock(15? and 20?)and heat shock(30?,35?,40? and 43?).The expression level of NbLeuHsp40,NbLeuHsp60 and NbLeuHsp90 of N.barkeri treated with high temperature(30°C)were significantly higher than controls. |
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