Transcriptome Analysis And Chitinase Gene Expression Patterns In Microdera Punctipennis Under Low Temperature Stress

Insects in Tenebrionidae have unique stress adaptations that allow them to survive temperature extremes. Microdera punctipennis（Coleoptera: Tenebrionidae） is an endemic beetle in the Gurbantunggut Desert and one of the “indicators of desertization”. We report here a gene expression profiling of Microdera punctipennis（Coleoptera: Tenebrionidae）, a beetle in desert region, to gain a global view of its environmental adaptations. In this dissertation, Illumina high-throughput sequencing technology was used to analyze the differentially expression unigenes in cold stress adult beetle, Microdera punctipennis. By high-throughput sequencing,we obtained abundant transcriptome information that could contribute to novel gene identification and genome research in desert beetles. Based on the transcriptome data, possible roles of the cold responsive genes and hydrolytic enzymes played in M. punctipennis were discussed. The main conclusions were as follows:（1） Transcriptome analysis of normal control group: a total of 48, 158, 004 reads were obtained by transcriptome sequencing in the normal temperature group, and the de novo assembly yielded 56, 348 unigenes with an average length of 666 bp. Based on similarity searches with a cut-off E-value of 10-5 against protein sequence databases, 41, 109 of the unigenes（about 72.96%） were matched to known proteins. A total of 8, 980 M. punctipennis unigenes were assigned COG classifications, and the BLAST2 GO annotation tool assigned 26, 332 of the unigenes to 1, 754 Gene Ontology terms. In addition 9, 986 of the unigenes were assigned to 283 KEGG pathways.（2） Transcriptome analysis of normal control group and cold induced group: the results of the transcriptome research showed that cold treatment group and normal group obtained 144, 777 unigenes with average length 463 bp. Among them, 51, 447 unigenes（35.54%） were annotated to the public protein database, however, there were large number of unigenes（64.46%） still function unknown. These sequences are a valuable resource for future studies of the desert beetle and other insects in Tenebrionidae. Transcriptome analysis based on Illumina paired-end sequencing is a powerful approach for gene discovery and molecular marker development for non-model species.（3） Analysis of differentially expression unigenes under low temperature stress: a total of 92, 010 unigenes were identified as cold-responsive unigenes in which 144, 777 unigenes. Gene Ontology annotation analysis showed 1, 231 up-regulated unigenes belonged to the hydrolase activity（GO: 0016787）. Gene ontology annotation analysis identified many cold-relevant categories, including “response to temperature stimulus”, “response to stress”, “response to heat”, “response to oxidative stress” and “thermotaxis”, etc. mitogen-activated protein kinase kinase 7（MKK7）, histidine decarboxylase（HDC）, molecular chaperone DnaJ（DNAJ）, heat shock protein 5（HSPA5/BIP）、neurofibromin 1（NF1） genes involved in response to temperature stimulus（GO-ID 9266）. Furthermore, RPKM data were validated by real-time PCR using a set of 13 unigenes, all of these unigenes behaved in accordance with the two expression methods. The dynamic expression changes reflect the integrative controlling and transcriptome regulation of the networks in the cold stress response of M. punctipennis. The biological processes involved in response to temperature stimulus and hydrolase activity might shed light on the molecular mechanisms related to cold tolerance in desert beetles and provide useful candidate genes for genetic improvement.（4） Preparation and titer assay of antiserum against chitinase MpCHT12: based on the transcriptome of M. punctipennis under cold stress conditions, we identified a chitinase gene（unigene ID NO.786seq1） named Mpcht12 which was highly induced by cold stress. The Mpcht12 gene was 837 bp with a 744 bp open reading frame encoding a peptide of 248 aa. Total RNA was extracted from the adult M. punctipennis, and then reverse transcribed to synthesize cDNA. Mpcht12 cDNA was amplified by RT-PCR, and inserted into pET30 a vector to construct the recombinant expression plasmid pET30a-Mpcht12. Fusion protein His-MpCHT12 was expressed in E.coli BL21 by IPTG induction. This fusion protein was used as antigen after recovered and purified by the SDS-PAGE gel slice and mixed with Freund’s adjuvant. In addition, eukaryotic expression plasmid pcDNA3-Mpcht12 was constructed and injected into mice from the tail vein by high-pressure injection. The transient expression of the pcDNA3-Mpcht12 in liver was detected by RT-PCR after 8h of the injection. For immunization, pcDNA3-Mpcht12 plasmid was used as DNA vaccine to inoculate mice for three times prior to the twice His-MpCHT12 protein boost immunization. After DNA immunization and the final immunization, antiserum was collected and identified by Western blotting for the specificity, and assayed by ELISA for the titer. RT-PCR results showed that after 8h of the tail vein high pressure injection, the eukaryotic expression plasmid pcDNA3-Mpcht12 was specifically expressed in mouse liver. Western blotting results with His-MpCHT12 as the antigen showed a specific band with the antiserum generated after three times of immunization with pcDNA3-Mpcht12 plasmid, and with the final antiserum after two times protein boost immunization with His-MpCHT12 fusion protein. ELISA assay showed that the titer for the final antiserum was over 1: 204800. High titer and specific antiserum against Mpcht12 from M. punctipennis was produced by using DNA prime-protein boost immunization strategy.（5） Expression pattern and function prediction of cold stress related gene Mpcht12: tissue expression pattern by real-time PCR and Immunohistochemistry revealed that Mpcht12 mRNA existed in foregut, carcass and mid gut, but mainly expressed in fat bady and hindgut, and protein expressed in fat bady and hindgut. Cold induction at 4oC for different times revealed that Mpcht12 expression started to increase after 1 h treatment, and then decreased at 5 h, but increased markedly after 7 h treatment, and then decreased at 9 h and 11 h treatment. A predicted protein interaction network consisting of 23 target genes was obtained by Search Tool for the Retrieval of Interacting Neighboring Genes/Proteins（STRING） software. N-acetyl-D-glucosamine kinase（XP₉72191）, beta-hexosaminidase B（XP₉75660）, beta-hexosaminidase subunit alpha-like（XP₉75658）, hexosaminidase A（XP₉75656）, fused lobes（FDl,NP₀01092296.1）, beta-N-acetylglucosaminidase（NAG2, NP₀01092298） and hexosaminidase 1（NAG1,XP₀08195558） were the top 7 genes with highest degrees of connection. The above results suggest that Mp CHT12 may be linked to changes in chitin metabolism and provide a new candidate gene for hypothesis-based investigations of the cold stress response.（6） Characterization and expression analysis of six chitinase genes: six chitinase genes from cold treated desert beetle Microdera punctipennis obtained by RNA-seq technology were characterized, and their expression patterns in different tissues and in response to cold were investigated. Multiple sequence alignment was carried out using ClustalW1.81 and Phylogenetic trees were generated by MEGA5. The expression patterns were studied by quantitative real-time PCR. These genes were assigned to three different chitinase groups. Almost all of them were highly expressed in midgut, some are expressed in fat body or hindgut.-4oC had stronger effect than 4oC in inducing chitinase expression. Chitinase genes from M. punctipennis exist in a big family. The tissue specific and cold inducible expressions suggest that the chitinases may have diverse functions and play roles in insect cold adaptation.