Construction and characterization of a cDNA library from wheat infected with Fusarium graminearum Fg 2

Abstract

Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

Keywords: 15 acetyl deoxynivalenol (15ADON); 3 acetyl deoxynivalenol (3ADON); Fusarium graminearum Fg2; Triticum aestivum; cDNA library.

Figure 1

(1.1%) Agarose gel electrophoresis of total RNA. Total RNA extracted from Fusarium-infected wheat spikes was fractioned. Lanes 1, 2, 3, 4, 5, 6, and 7 represent samples collected 6, 12, 24, 36, 48, 72hr, and 6 days after-inoculation, respectively. One microliter of RNA was loaded for each sample.

Figure 2

A 1.1% agarose gel electrophoresis of Poly(A)⁺ RNA. A uniform smearing pattern of the Poly(A)⁺ RNA indicates a high mRNA quality purified from total RNA. M: RNA ladder; Line 1: Poly(A)⁺ RNA (0.2 μg).

Figure 3

A 1.1% agarose gel electrophoresis of ds cDNA after PCR. One μg (3 μL) of Poly(A)⁺ RNA was used as RNA template with the indicated primers in a first-strand synthesis. 11 μL of ss cDNA served as a template for primer-extension based, second-strand synthesis using 3 thermal cycles. Lane M: HyperLadder marker (5 μL). Lane 1: 5 μL sample of the ds cDNA product showing a smear ranging from 0.2 to 3 kb.

Figure 4

A 1.1% agarose gel electrophoresis of size-fractionated cDNA for removal of small oligonucleotides. Single-drop fractions (15 fractions, 35 μL each) were collected in separated tubes, and 3 μL of each fraction was electrophorised on 1.1% agarose gel at 150 V for 10 min. The lightest lanes (circled numbers 6–11) indicating cDNA peaks were collected for ligationstep. M: HyberLadder marker; Lanes 1–15 are cDNA size-fractionated samples.

Figure 5

(A) The titer of unamplified library. (B) Blue and white screening of the unamplified library. Arrows indicate blue plaques. The two plates (A, B) were inoculated with a 10-fold dilution of packaged phage. Plaques were approximately 1 mm in diameter. (C) The titer of amplified library. (D) Blue and white screening of the amplified library. Arrows indicate blue plaques. The two plates (C, D) were inoculated with a 1 × 10⁴ dilution of phage lysate. Bacteriophage: λ lcI857 Sam7. Host: Escherichia coli LE392MP.

Figure 5

(A) The titer of unamplified library. (B) Blue and white screening of the unamplified library. Arrows indicate blue plaques. The two plates (A, B) were inoculated with a 10-fold dilution of packaged phage. Plaques were approximately 1 mm in diameter. (C) The titer of amplified library. (D) Blue and white screening of the amplified library. Arrows indicate blue plaques. The two plates (C, D) were inoculated with a 1 × 10⁴ dilution of phage lysate. Bacteriophage: λ lcI857 Sam7. Host: Escherichia coli LE392MP.

Figure 6

A 1.1% agrose gel electrophoresis of PCR products of cDNA inserts selected randomly (165 plaques) from the amplified cDNA library. Lane M: 1 kb Plus DNA marker (Invitrogen); 31–60: A part of plaques selected randomly and insert-amplified by PCR.

Figure 7

Flow diagram of the cDNA library construction.