Cloning And Characterization Of The Immune-Related Genes And Tubulin Genes From The Small Brown Planthopper

The small brown planthopper, Laodelphax striatellus, is the vector of Rice Stripe Virus (RSV) and Rice Black Streaked Dwarf Virus (RBSDV). To understand the molecular regulation of the immune response in Laodelphax striatellus, the relevant subset of differentially expressed genes of interest must be identified, cloned and studied in detail. In this study, suppression subtractive hybridization, a PCR-based method for cDNA subtraction was performed to identify mRNAs from the body of immunized larvae that are not present (or present at a low level) in control larvae.Two subtracted cDNA libraries enriched in immune-inducible genes were constructed. 44 and 13 positive clones were selected from E.coli –immunized subtracted library and RSV-infected subtracted library, respectively. Sequence analysis revealed that 22 differentially expressed gene fragments, representing 13 distinct genes or gene families, had sequence identities or similarities to previously characterized genes, some of which have been confirmed to be involved in innate immunity. The rest of differentially expressed gene fragments, about 50% of the library, showed no significant similarity to any known genes. Northern Blot was performed to determine the expression pattern of mucin gene that was obtained from RSV-infected subtracted library. The result showed that the transcript of mucin could only be detected in RSV-infected planthopper. Therefore, as a kind of rapid and convenient method, SSH could be applied in cloning immune-related genes in L. striatellus.RACE (Rapid Amplification of cDNA Ends) was carried out and 3' full length was obtained. We cloned this fragment into expression vector and yielded corresponding peptides.Microtubules are framework proteins that decide growth and development of plant hoppers. It's reported that microtubules may be the target of control. The mRNA isolated from adult Laodelphax striatellus was reverse transcribed. Using degenerate primers designed according to the α- and β-tubulin sequences of Drosophila melanogaster and Bombyx mori, cDNA fragments were amplified from Laodelphax striatellus mRNA. To obtain the full-length cDNA, 3'and 5' RACE were carried out by 3' and 5' RACE System for Rapid Amplification of cDNA Ends (RACE). Two isotypes of α-tubulins and two isotypes of β-tubulins were found in Laodelphax striatellus. The α- tubulins were designated LSTUA1 and LSTUA2 while the β-tubulins were designated LSTUB2 and LSTUB3. The nucleotide sequences obtained were deposited in Gene Bank with the following accession numbers: AY508717 for LSTUA1, AY550922 for LSTUA2, AY479977 for LSTUB2, and AY334072 for LSTUB3.We constructed phylogentic tree using these tubulin genes plus those that we obtained from NCBI Gene Bank by BLAST. Results show that these tubulin genes, LSTUA1, LSTUA2, LSTUB2 and LSTUB3 could be classified into different subfamily. They share high similarity with tubulin genes from other insect. Alignment analyses suggest tubulin genes from plant hoppers contain corresponding characteristic sequences.Southern blot data indicated that LSTUB3 is present as a single copy locus in L. striatellus genome. Northern analysis demonstrated certain stage specific expression pattern of LSTUB3. The LSTUB3 was expressed in vitro in Escherichia coli BL21 and a 76-kDa fusion protein was detected by Western analysis.