Cloning, MRNA Expression, And Feeding RNAi Analysis Of Chitin Synthase And Chitin Deacetylase From Cotton Bollworm, Helicoverpa Armigera

Helicoverpa armigera is a major pest in cotton fields. Application of chemical pesticides and Bt crops in controlling the pests cause resistance to insecticides. RNA interference(RNAi) holds great potential as a new measure for pest management because of its high specificity and efficiency. The target gene is the key element for silencing effect. In insect, chitin is a vital component of the cuticle and the peritrophic membrane, and it is absent invertebrates and plants. Besides, chitin metabolism is crucial for growth and development. Chitin metabolism related genes have attracted much research interest because of their potential as specific target for RNAi-based pest control.In this research, chitin synthase and chitin deacetylase, two chitin metabolism related genes of H. armigera, were cloned, and the expression levels of those genes in different developmental stages and tissues were analyzed by real-time quantitative PCR(RT-q PCR). Furthermore, RNAi phenotypics were observed of feeding RNAi experiments. Hence, it provides a theoretical basis for effective H. armigera control based on RNAi. The contents are as follows:1. Molecular cloning and expression analysis of chitin synthase genes in H. armigeraHa CHS1 and Ha CHS2, two chitin synthase encoding genes, were first identified from H. armigera. Ha CHS1 had two alternative splicing variants, Ha CHS1 A and Ha CHS1 B. The two variants differed only in one exon consisting of 177 nucleotides and encoding 59 amino acid residues. The nucleotide and amino acid sequences within this alternative splicing region were 64% and 69% identical between the two variants. The ORF of Ha CHS1 c DNA was 4698 bp in length, encoding 1565 amino acid residues. The ORF of Ha CHS2 c DNA was 4587 bp in length, encoding a protein of 1528 amino acid residues. The expression pattern in different developmental stages and tissues of Ha CHS in H. armigera was measured by RT-q PCR, and the results revealed that the expression level of Ha CHS2 was highest in the foregut, lower in the midgut and hindgut, and lowest in the malpighian tubules, integument, fat body. However, Ha CHS1 A was highly expressed in the integument, and relatively weakly expressed in the hindgut and the foregut, with only trace levels in the malpighian tubules, midgut, fat body. whereas Ha CHS1 B showed nearly the same m RNA expression level in the hindgut, foregut and integument. The m RNA expression levels of Ha CHS1 A and Ha CHS1 B were mainly detected in larva stage, pupa stage, and adult stage, whereas Ha CHS2 was predominantly expressed in egg stage and larva stage.2. Molecular cloning and expression analysis of chitin deacetylase genes in H. armigeraFour chitin deacetylase encoding genes, including Ha CDA1, Ha CDA2, Ha CDA5 a and Ha CDA5 b, were cloned from H. armigera. Ha CDA2 was a new chitin deacetylase encoding gene, with an intact open reading frame(ORF) of 1611 nucleotides. The encoded protein was 536 amino acids in length with a predicted molecular weight(MW) of approximately 60.78 k Da. The predicted isoelectric point(p I) of Ha CDA2 was 5.31. Sequence alignment and phylogenetic analysis of the putative protein revealed that Ha CDA2 belongs to CDA group I. The highest expression level of Ha CDA2 was detected in fat body among issues tested in the fifth instar larvae using RT-q PCR method. Ha CDA2 gene was expressed mainly in larva stage and pupa stage.3. Phenotypic observation and expression analysis of feeding RNAi experiments.Escherichia coli strain HTI15 was engineered to express ds RNA targeting Ha CHS and Ha CDA and used for feeding RNAi experiments. The RT-q PCR results showed that m RNA levels of all target genes were reduced compared to ds EGFP control group on 48 h after continuous feeding of engineered bacteria. The expression levels of ds Ha CHS1, ds Ha CHS1 A, ds Ha CHS1 B, ds Ha CHS2, ds Ha CDA1, ds Ha CDA2, ds Ha CDA5 a and ds Ha CDA5 b treated groups were reduced to 73.1%, 23.8%, 14.0%, 8.3%, 82.9%, 79%, 38%, 51%, respectively. Generally speaking, interfering efficiency in non-gut genes were significantly lower than that in gut genes. Feeding experiment indicated that interfering of Ha CHS1, Ha CHS1 A, Ha CHS1 B, Ha CHS2, Ha CDA5 a and Ha CDA5 b but not Ha CDA1 and Ha CDA2 led to inhibition of larvae growth and development. Furthermore, an enhanced resistance of cotton leaf to larvae was observed after feeding with leaves coated with engineered bacteria expressing ds RNA of Ha CHS1, Ha CHS1 A, Ha CHS1 B, Ha CHS2, Ha CDA5 a and Ha CDA5 b genes.This study provides a theoretical basis for effective H. armigera control based on the target genes of Ha CHS, Ha CDA5 a and Ha CDA5.