E conserved regions characteristic of PL3 pectate lyases. Very highly conserved

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E conserved regions characteristic of PL3 pectate lyases. Very highly conserved charged residues are indicated beneath the alignment by a number symbol (#), while an asterisk (*) indicates conserved cysteine residues. The positions of the intron in AA-PEL-1 and AA-PEL-2 are indicated by A1 and A2 respectively, that in BXPEL-1/2 and BM-PEL-1/2 by B, and those in MI-ENG-1 by Mi1. Triangles and diamonds represent phase 0 and 1 introns, respectively.eng-1 and Aa-pel-1 specifically hybridized with the transcripts in the esophageal gland cells of A. avenae (Figs. 9A and Staurosporine – https://www.medchemexpress.com/Staurosporine.html 9C). Staining was observed in juvenile and adult nematodes rather than being restricted to a specific lifestages. No hybridisation was observed with the control (sense) cDNA probes of Aa-eng-1 or Aa-pel-1 (Figs. 9B and 9D).Page 13 of(page number not for citation purposes)BMC Genomics 2009, 10:http://www.biomedcentral.com/1471-2164/10/As a part of the complex process of parasitism, a wide range of plant parasitic nematodes use endogenous -1,4endoglucanases and pectate lyases to degrade two abundant constituents of the plant cell wall and thus facilitate their migration through host tissues. The presence of signal peptides in the deduced amino acid sequences of the endoglucanases and pectate lyase from A. avenae coupled with their expression in the esophageal glands suggest that both enzymes have a similar role in A. avenae. The presence of such genes in A. avenae suggests that this nematode can enter and migrate through plant tissues and may also be able to feed on plant cell contents. This is backed up by the observation that A. avenae is known to feed on plant tissue in culture [13,14]. A. avenae may therefore have a wide ranging diet that includes fungi and plant tissues. This, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 – https://www.ncbi.nlm.nih.gov/pubmed/6833145 coupled with the position of this nematode as a basal member of a clade that includes a wide range of plant parasitic nematodes, provides further support for the idea that plant parasitism has evolved from fungal feeding and suggests that A. avenae, may be a very primitive plant feeder.by three washes with 0.5?PBST [70]. Nematodes were stored at -80 until use.Isolation of total RNA, cDNA synthesis and cDNA library construction Total RNA from mixed stage A. avenae was isolated using Sepasol (Nakalai). Analysis of the total RNA on a denaturing agarose gel showed a smear from 50 to 3,000 bp with two distinct bands of ribosomal RNA. Poly(A)+ RNA was extracted from total RNA using a FastTrack?MAG micro mRNA Isolation Kit (Invitrogen). cDNA was synthesized using the SMART PCR cDNA amplification method (Clontech) with a NotI oligo-dT primer (5′ AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT). SalI/SmaI adaptors (Takara) were added to double stranded cDNA which was digested with NotI and size fractionated using a cDNA Size Fractionation Column to remove small cDNA (< 500 bp) (Invitrogen). The appropriate fractions containing cDNA were pooled and ethanol precipitated. Inserts were directionally cloned into NotI and SalI sites of the pSPORT1 vector, and transformed into Escherichia coli DH5 cells. The cDNA library was designated as Aamk. EST generation Individual transformants (n = 5,472) from the plasmid library were picked into 96 well plates containing 0.5 ml of LB medium containing 100 g/ml ampicillin. Plates were incubated overnight at 37 . A small aliquot of each culture was stored at -80 after being mixed with same volume of 25 glycerol in LB. Plasmid DNA was isolated and purified using FB glass fiber.