Study On Important Genes In The Biosynthetic And Metabolic Pathways Of Chitin In Panonychus Citri

Chitin plays a key role in insect (mite) growth and development. The growth and development of insect (mite) need precisely control of the synthesis and degradation of chitin. Chitin is widely distributed in protozoan, nematodes. insect and shellfish, but not exists in plant and mammal. Chitin biosynthesis and metabolic pathway is a hot spot in the area of insect (mite) growth and development. The citrus red mite Panonychus citri (MeGregor) is an important pest worldwide that can devastates more than 112 plant species. In China, this mite invades all citrus planting areas and has become one of the most serious mites in citrus orchard. Recent population outbreaks may be attributed to the intensive management of the orchard and the vast irrational use of acaricides, resulting in decreased the natural enemy of P. citri. In addition, it is difficult to manage because of its ability to develop resistance to acaricides. Therefore, it is urgent to find a new pest management strategy to instead of the traditional protection methods. Our study focused on the growth and development pattern of P. citri. We analyzed the molecular characteristics and the function of important gene in the biosynthesis and metabolic pathway based on the P. citri transcriptome database. The main content are as follows:1. Monitoring of resistance to acaricides in different field populations in Sichuan and ChongqingUsing a modified leaf dip method, the resistance levels of eight P. citri geographic populations (Sensitive Strain, Beibei, Changshou, Wanzhou, Fengjie, Wulong, Anyue, Luzhou) samples in Sichuan and Chongqing to five acaricides (Abamectin, Pyridaben, Fenpropathrin, Bifenthrin, Cyflumetofen) were measured. The result showed that Pyridaben exhibited the hightest acaricides activity among the five acaricides, while Cyflumetofen, Abamectin and Fenpropathrin showed a less susceptibility to the field populations. However, the Bifenthrin exhibited the hightest resistance level on eight P. citri field populations in Sichuan and Chongqing. In addition, the acaricides activity of five acaricides to the eight field population are ranked as Pyridaben> Cyflumetofen> Abamectin> Fenpropathrin> Bifenthrin.2. Molecular cloning and sequence analysis of chitin synthase 1 gene from P. citriBased on the transcriptome data, the full length sequence of chitin synthase 1 (PcCHS1) gene (GenBank number:KF241748), was cloned from P. citri using RT-PCR and RACE techniques. The alternative splicing variant, PcCHS1b, was identified from P. citri using gDNA as the amplification template:, which means it have no alternative splicing. The cDNA of PcCHSl contained an open reading frame (ORF) of 4,305 bp, encoding 1,535 amino acid residues. Multiple protein alignments of Tetranychus urticae, Metaseiulus occidentalis and insects chitin synthase, it showed that PcCHSl have three conservative domains:domain A (N-terminal domain) with 11 transmembrane helices; domain B (a high conserved catalytic domain) with two signature motifs, EDR and QRRRW; and domain C (C-terminal domain) with 7 transmembrane helices and a signature motif TWGTR, which was assumed to play a critical role in chitin translocation. A phylogenetic analysis showed that CHS1 gene from T. urticae and P. citri clustered into the CHS1 family and seemed to share a single clade. This result indicated that similar physiological functions and evolutionary relationship may exist between the CHS1 in T. urticae and P. citri. During P. citri growth and development stage, PcCHSl was highly expressed during the eggs stage, while lowly expressed in the adult stage. Exposure of P. citri larvae to DFB for 6 h, the relative expression level of PcCHS1 was up-regulated significantly compared to the control. The above-mentioned results indicate that exposure to DFB result in inhibiting the chitin biosynthesis, increased CHS1 expression may indicate the existence of a feedback regulatory mechanism that compensates for the enzymes content.Chitin content were determined against different developmental stages and after DFB treatment of P. citri via the 96-well microplate reader. The total chitin content appear a tendency of increased first and then reduced in the developmental stages. The chitin content reached its highest level at larvae and lowest level at adult stage. Exposure of P. citri to DFB caused significant reduce of the chitin content. After treatment with DFB at low-lethal concentration (LC10), the chitin content of P. citri was half compared to control group. While treatment with DFB at sub-lethal concentration (LC30) and median lethal concentration (LC50), the chitin content raised a little but still lower than control.3. Molecular cloning and functional analysis of three chitinase genes in P. citriIn this study, we cloned and characterized three full length cDNA sequences of chitinase genes in P. citri. Gene names and GenBank accession numbers are as follow: PcCht1 (KJ528304). PcCht2 (KP319018) and PcCht4 (KJ528305), respectively. The cDNA of PcChtl contains an open reading frame (ORF) of 1,695 bp, encoding 564 amino acid residues. The PcCht1 genome sequence was the same compared with PcCht1 cDNA sequence. It has no introns but only one exon. The cDNA of PcCht2 contains an open reading frame (ORF) of 1.935 bp, encoding 644 amino acid residues. The cDNA of PcCht4 contains an open reading frame (ORF) of 1,587 bp, encoding 528 amino acid residues. The PcCht4 genome has eight exons and seven introns.All of the seven introns have the GT-AG signature sequence. The predicted molecular of PcCht1, PcCht2 and PcCht4 are 62.7,70.7 and 59.9 kDa and the isoelectric point are 5.6,8.5 and 6.0, respectively. Phylogenetic analysis showed that chitinase genes from P. citri and T. urticae are clustered together.RT-qPCR analysis showed that PcCht1, PcCht1 and PcCht4 wrere mainly expressed during larvae stage, suggesting that these three genes may play key roles in the biological processes in developmental and growth stages in P. citri. DFB induction significantly increased the PcCht1, PcCht2 and PcCht4 expression levels. The artificial feeding RNA interference system was established successfully. Feeding of dsRNA of PcCht1 with larvae significantly reduced its expression level of PcCht1 with 55% silence efficiency and showed abnormal phenotypes. The mortality rate is reached 60.9% and molt rate is only 10.9% after 48 h. However silencing of PcCht4 has no morphology.In summary, we estimated the resistance monitoring of different field populations of P. citri in Sichuan and Chongqing. One chitin synthase and three chitinase full length cDNAs were cloned and characterized based on the P. citri transcriptome databas. Transcription profiles of these genes were determined in different developmental stages and acaricides induction. Chitin content was also measured in different developmental stages and acaricides induction using the 96- well microplate reader. Furthermore, artificial feeding RNA interference system was also successfully established. RNAi technology was performed to investigate the genes function. Results of our research enriched the information of chitin biosynthesis and metabolism of these genes and will provide basic data for studying chitin synthesis pathway.