Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.

Made available in DSpace on 2018-01-04T23:23:41Z (GMT). No. of bitstreams: 1 journal.pone.0189600.pdf: 7131320 bytes, checksum: ece3da5d8a008843e58701868100618d (MD5) Previous issue date: 2018-01-04bitstream/item/170309/1/journal.pone.0189600.pd

Protein SDS-PAGE of purified inducible LAP from pigeon pea leaves.

<p>The crude leaf extract and ion-exchange fraction were loaded on 12% SDS-PAGE and separated protein bands were visualized by staining with CBBR-250. Lane 1, Molecular weight markers, Lane 2, crude leaf extract and Lane 3, purified LAP from ion-exchange fraction.</p

Interactions of pigeon pea inducible LAP with <i>H. armigera</i> gut proteinases (HGPs).

<p>To see the direct effect of inducible LAP on <i>H. armigera</i> gut serine protease activity present experiment was undertaken. A- Detection of aminopeptidase activity isoforms from the midgut extract of <i>H. armigera</i> by incubation with inducible LAP. Midgut extract (40 µg protein) of larvae fed on control diet was pre-incubated with pigeon pea LAP (40 µg protein) for at 37°C for 30 min and aminopeptidase activity isoforms in the mixture were electrophoretically visualized. Lane 1, crude midgut extract, lane 2, crude midgut extract+pigeon pea LAP and lane 3, pigeon pea LAP. B- Detection of proteinase activity isoforms in <i>H armigera</i> midgut extract when incubated with pigeon pea inducible LAP. Midgut extract (40 µg protein) of larvae fed on control diet was pre-incubated with pigeon pea LAP (40 µg protein) at 37°C for 30 min and proteinase activity isoforms in the mixture were visualized by gelatin reverse zymography. Lane 1, crude midgut extract, lane 2, crude midgut extract+pigeon pea LAP and lane 3, pigeon pea LAP.C- Determination of aminopeptidase, trypsin and chymotrypsin activities in <i>H armigera</i> midgut extract when incubated with pigeon pea inducible LAP. Midgut extract (40 µg protein) of larvae fed on control diet was pre-incubated with pigeon pea LAP (40 µg protein) at 37°C for 30 min and proteinase activities were determined by using selective chromogenic substrates such as L<i>p</i>NA, BA<i>p</i>NA and SAAPF<i>p</i>NA respectively for. The respective reactions were started at zero time and continued up 30 min (for aminopeptidase) and 1 h (for trypsin and chymotrypsin) and terminated by 30% acetic acid. The released <i>p</i>-nitroaniline from the substrates by the action of respective protease was measured at 410 nm. The assay was carried out in triplicate.</p

Determination of activities of aminopeptidase, trypsin and chymotrypsin in the midgut of <i>H. armigera</i> larvae fed on artificial diet and diet containing LAP.

<p>The spectrophotometric assay was carried out at 37°C using selective substrates viz. L<i>p</i>NA for aminopeptidase, BA<i>p</i>NA for trypsin and SAAPF<i>p</i>NA for chymotrypsin. Rate of liberated of <i>p</i>-nitroaniline in the reaction was measured at 410 nm. The experiment was performed three times with three biological replicates.</p

In-gel visualization of aminopeptidase and proteinase activity isoforms in the midgut of <i>H. armigera</i> larvae fed on artificial diet and diet incorporated with plant LAP.

<p>A- Midgut extracts (100 µg of proteins each) were resolved on 8% native discontinuous polyacrylamide gels and aminopeptidase activities were stained with 1- naphthylamine by diazotizing the released <i>p</i>-nitroaniline from L<i>p</i>NA with sodium nitrite. Three pink bands of the aminopeptidase activity were observed and these bands were designated as AP1 to AP3. No difference in aminopeptidase activity band pattern was observed between larvae fed on artificial diet and diet supplemented with LAP. B- Midgut extracts (40 µg of proteins each) were resolved on 8% native discontinuous polyacrylamide gels and proteinase activity isoforms were detected using gelatin reverse zymography. The proteinase activity bands were observed white against blue background. These bands were designated as HGP1 to HGP10. The activity of proteinase isoforms was significantly decreased in the midgut extracts of larvae reared on diet supplemented with pigeon pea LAP.</p

Growth and survival rate of <i>H. armigera</i> larvae on artificial diet and diet containing pigeon pea inducible LAP.

<p>A- Survival rate of <i>Helicoverpa armigera</i> on a control diet versus test diet containing pigeon pea inducible LAP. The neonates were fed upon diet containing pigeon pea inducible LAP. The feeding analysis was carried out till 12 days and rate of survival was recorded every 3 day. Data were analyzed by a nonparametric chi-square test to determine significant differences in the percentage survival of <i>H. armigera</i> fed on the test diet (<i>n</i> = 30 individuals per test, three replicates). The highest survival rate was recorded on the control diet, whereas the lowest survival rate was recorded on the diet containing plant LAP. B- Development of <i>H. armigera</i> fed on control and test diet. Larvae fed on control diet showing normal growth, while larvae fed on plant LAP showing retarded or stunted growth. The aminopeptidase denatured by heating was used as+ve control on which the larval growth was found similar to as of control.</p

Substrate preferences of aminopeptidases of <i>H</i>. <i>armigera</i> fed diet with and without LAP.

<p>1 unit (U) = one micromole of <i>p</i>-nitroaniline released/mg of protein/min at 37°C.</p

Evaluation of larval mass of <i>H. armigera</i> larvae fed on control diet and diet containing plant LAP and denatured plant LAP.

<p>The experiment was performed in three replicates, and each replicate contains thirty larvae. Significant differences in larval mass were calculated by Student <i>t</i> tests. Error bars indicate ± SD. Significant difference in the mass of larvae fed on control and test diet was observed on day 9 (<i>P</i><0.01) and days 12 and 15 (<i>P</i><0.001) of feeding. Larval mass gain was adversely affected to feeding on inducible LAP.</p