Molecular Characterzation And Functional Study On Chitin Synthase Genes From Locusta Migratoria Manilensis (Meyen)

Locusta migratoria manilensis (Meyen) is an important agricultural pest and the plague of locusts poses a serious threat to agriculture in China. Organophosphate (OP) insecticides are still the major measures to control the locusts. The frequent and excessive use of OPs can cause a series of problems, such as the resistance to OPs, contaminating environment and making harm to non-target organisms. Since chitin is widely found in arthropods, fungi and nematodes and absent in higher animals and human, chitin metabolism has been recognized as a novel target for developing safe and effective insecticides. In this paper, two chitin synthases of L. migratoria manilensis, the key enzymes in chitin synthesis, were characterized in molecular level. It is expected to be helpful for designing new pesticides and pest management. The contents are as follows:1. Molecular cloning of chitin synthase genes from L. migratoria manilensisThree CHS fragments were first obtained from LocustDB. Pairs of gene-specific and degenerate primers were designed to obtain overlapping PCR products. A phylogenetic tree of insect chitin synthases was generated, which shows that insect chitin synthases are grouped into two classes, CHS1 and CHS2. The chitin synthase genes we obtained were named LmCHSl and LmCHS2, respectively. Each cDNA of the two variants (LmCHS1A and LmCHS1B) consists of 5,116 nucleotides that include a 4,728-nucleotide open reading frame encoding 1,576 amino acid residues and a 67- or 321- nucleotide for 5'- or 3'- non coding region, respectively. The two variants differ only in one exon consisting of 177 nucleotides. The amino acid sequences within this alternative splicing region are 75% identical. The cDNA of LmCHS2 consists of 5,018- nucleotides, including an ORF of 4,569 nucleotides that encode 1,523 amino acid residues and a 76- or 373- nucleotide for 5'- or 3'- non coding region, respectively. The calculated Mr of LmCHS1 and LmCHS2 proteins are about 179 kDa and 174 kDa and pI are about 6.65 and 6.89, respectively.2. Tissue-specific and developmental expression patterns of LmCHSRT-PCR and Real-time PCR were carried out using gene-specific primers to analyze the expression patterns of LmCHS in different tissues and developmental stages of L. migratoria manilensis. The results indicated that LmCHS1 was predominantly expressed in integument and trachea. LmCHSIA was highly expressed in integument, whereas LmCHSIB was mainly expressed in the trachea, and LmCHS2 was specifically expressed in midgut and gastric caeca, which suggest LmCHS1 mainly catalyse chitin synthesis of integument and trachea and LmCHS2 is responsible for chitin synthesis of midgut and gastric caeca. LmCHS1 was consistently expressed in all life stages. The expression level of LmCHS1 was lowest in eggs but highest in adults. The expression pattern of LmCHS1A was similar to that of LmCHS1. LmCHSIB showed a low expression level in eggs, the 4th and 5th instar nymphs and relatively high expression levels in the 1st,2nd,3rd instar nymphs and adults. There was no detecable expression in eggs for LmCHS2, however, the expression gradually increased from the 1st to 3rd instar nymphs, and reached the highest in 3rd instar nymph, then keeped this expression level to adults. The results suggested that chitin synthases are essential for the growth and development of L. migratoria manilensis.3. Functional analysis of chitin synthase genes from L. migratoria manilensisTo further explore biological functions of LmCHS of L. migratoria manilensis, RNAi was performed by injecting dsRNA to the 2nd instar nymphs. The nymphs injected with LmCHS1 dsRNA displayed slowly development, the ecdysis time longer compared to the control, and can not detach old cuticle and molt to the next stadium. The terminal phenotypes resulted from the injection of LmCHS1A dsRNA were similar to those from the injection of LmCHSl dsRNA. However, nymphs injected with LmCHSIB dsRNA exhibited crimpled cuticle phenotype, which was different from those obtained with injection of dsRNA for LmCHS1 or LmCHS1A. Nymphs injected with LmCHS2 dsRNA decreased feeding dramatically and finally died. Consequentlly,95,88,51 and 95% of mortalities were observed in the locusts injected with the LmCHS1, LmCHS1A, LmCHS1B and LmCHS2 dsRNA, respectively, which were significantly higher than those in buffer injection (5%) and dsGFP injection (0%). Our RNAi-based silencing method resulted in a dramatic reduction in the levels of the corresponding mRNA in the locust nymphs injected with dsRNA for LmCHS. All these results indicated that chitin synthases play an important role in the growth and development of L. migratoria manilensis, and gene silencing can result in the development of locust blocked and even death.