High-amylose wheat generated by RNA interference improves indices of large-bowel health in rats

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Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
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Supporting Figure 5

Fig. 5. Schematic representation of the hp-SBEIIa construct generated for wheat and barley transformation. Fragment I is comprised of sequence from nucleotide positions 96-635 of wheat SBEIIa cDNA (GenBank accession no. Y11282) corresponding to exons 1, 2, and 3 of the wheat SBEIIa gene, wSBEII-DA1 (1), fragment II is comprised of sequence corresponding to intron 3 of wSBEII-DA1 and fragment 3 the same sequence as in fragment I but in the antisense orientation. For the hp-SBEIIb construct, fragment I is comprised of a sequence from nucleotide positions 77-604 of wheat SBEIIb cDNA (A.R., unpublished work) corresponding to exons 1, 2, and 3 of wheat SBEIIb gene, wSBEII-DB1 (A.R., unpublished work), fragment II is comprised of sequence corresponding to intron 3 of wSBEII-DB1, and fragment III the same sequence as fragment I but in the antisense orientation.

1. Rahman, S., Regina, A., Li, Z., Mukai, Y., Yamamoto, M., Kosar-Hashemi, B., Abrahams, S. & Morell, M. K. (2001) Plant Physiol. 125, 1314-1324.

Supporting Figure 6

Fig. 6. Identity of (a) wheat SBEIIa cDNA to hp-SBEIIa sequence and (b) wheat SBEIIb cDNA to hp-SBEIIa sequence generated by the software COMPARE of the GCG package. Regions of 21 bp identity are shown as vertical lines.

Supporting Figure 7

Fig. 7. Northern blot analysis of the expression of SBEIIa and SBEIIb mRNA in wheat transgenic lines. A shows Northern blotting of RNA extracted from endosperm 15 days post-anthesis from the SBEIIa transgenic line, 069 (lane 1), and the SBEIIb transgenic line, 008 (lane 2), probed with SBEIIa-specific probe targeting exons 1, 2, and 3 of the SBEIIa gene. A positive hybridization signal was observed in line 008 and not in line 069. B shows Northern blotting of mRNA extracted from the SBEIIa transgenic line, 069 (lane 1), and the SBEIIb transgenic line, 008 (lane 2), probed with SBEIIb specific probe targeting the exons1, 2, and 3 of SBEIIb gene. Only the line 069 showed positive hybridization signal using this probe.

Supporting Text

Wheat Transformation Vector. For the SBEIIa construct, three fragments of DNA containing sequence from the wheat SBEIIa gene, wSBEII- DA1 (GenBank accession no. AF338431) were amplified by PCR. These are fragment I, comprising the sequence from nucleotide positions 96-635 of wheat SBEIIa cDNA (GenBank accession no. Y11282) corresponding to exons 1, 2, and 3 of wSBEII-DA1 with the EcoRI and KpnI restriction sites on either side; fragment II, amplified from wSBEII-DA1, comprising the sequence from nucleotide positions 2220-2731, corresponding to the intron 3 region with the KpnI and SacI restriction sites on either side; and fragment III, comprising the same sequence as fragment I but with flanking restriction enzymes SacI and BamHI. For the SBEIIb construct, three fragments of DNA from the wheat SBEIIb gene, wSBEII-DB1 (1), were amplified by PCR. These are fragment I, comprising the sequence from the nucleotide position 77-604 of wheat SBEIIb cDNA (1) corresponding to exons 1, 2, and 3 of wSBEII-DB1, with the EcoR1 and KpnI restriction sites on either side; fragment II, amplified from wSBEII-DB1 comprising the sequence from nucleotide positions 1770-2364 corresponding to the intron 3 region with the KpnI and SacI restriction sites on either side; and fragment III, comprising the same sequence as fragment I but with flanking restriction sites SacI and BamHI. Fragments I, II, and III of each construct were then ligated so that the sequence of fragment III was ligated to fragment II in the antisense orientation relative to fragment I. The respective ligated sequence was introduced into an intermediate vector pDV03000 containing a high-molecular-weight glutenin (HMWG) promoter sequence from wheat and the terminator sequence of nopaline synthase (nos3') gene from Agrobacterium to generate SBEIIa (pDV03-IIa) and SBEIIb (pDV03-IIb) intermediate vectors. Each of the expression cassettes was then cut out from pDV03-IIa and pDV03-IIb with the restriction enzyme XhoI and inserted into the binary transformation vectors pGB53 and pBIOS340 to generate hp-SBEIIa and hp-SBEIIb constructs. pGB53 was created from pSB11 by the introduction of the gene encoding asulam resistance (su1) driven by the rice actin promoter, leaving a unique XhoI site adjacent to the right T-DNA border for the introduction of a gene of interest. Similarly, pBIOS340 was created from pSB1 by the introduction of an npt II gene encoding kanamycin and geneticin resistance, driven by the rice actin promoter, again leaving a unique XhoI site adjacent to the right border.

Extraction of Starch. The endosperm half of a single grain was crushed gently and soaked in 1 ml of deionized water at 4°C overnight. The softened endosperm tissues were ground with a small plastic pestle, removing the ball of gluten while grinding. Crude starch was recovered by passing the suspension through a 65-mm nylon mesh. After centrifugation for 3 min at 13,000 ´ g at room temperature, the supernatant was removed. The sedimented starch was washed three times with distilled water and once with acetone, each time vortexing thoroughly, centrifuging at 13, 000 ´ g for 3 min and removing the supernatant. Starch thus purified was dried at room temperature overnight.

Estimation of Amylose Content by Sepharose CL-2B Gel Filtration. In this method, the starch molecules are separated by the Sepharose CL-2B column based on their molecular weight. The separated fractions of starch were assayed by using the Starch Assay Kit (Sigma). Approximately 10 mg of starch was dissolved in 3.0 ml of 1 M NaOH (degassed) by incubation at 37°C for 30 min. The starch solution was centrifuged for 15 min to spin down the undissolved components. The supernatant was loaded onto a Sepharose CL-2B column at a pump speed of 1 ml/min. The column was run by using 10 mM NaOH buffer. Fifty fractions were collected in 10-ml tubes with 2.5 ml per tube. The pH of fractions 9-50 was adjusted to 4.5 with 35 ml of 1 M HCl. An aliquot (250 ml) of the starch sample was transferred into an Eppendorf tube followed by the addition of 250 ml of starch reagent (starch assay kit, Sigma). Along with the starch samples, four different controls were also set up: (i) a starch assay reagent blank containing only starch reagent (250 ml) and water (250 ml), (ii) a glucose assay reagent blank containing only 500 ml of water, (iii) a sample blank containing only 250 ml of starch sample and 250 ml of water, and (iv) a sample test containing only 250 ml of starch reagent and 250 ml of starch sample. The starch samples and the controls were incubated at 60°C for 60 min. After incubation, 200 ml each of the samples and the controls was transferred to a new Eppendorf tube followed by addition of 1 ml of glucose reagent (starch assay kit, Sigma) and incubation at 37°C for 30 min. The absorbance of the samples and controls was read at 340 nm. The quantity of starch (mg) in each fraction was calculated based on the equation given in the kit. The chromatogram reveals two peaks of elution. Amylose content is estimated as a percentage of the total amount of starch within the second peak of elution to the total amount of starch within both the peaks.

Sampling and Analytical Procedures. Rats were anaesthetized with halothane; the abdominal cavity opened; and cecal and colonic contents collected, weighed, and stored at -20°C until analysis. After removal of digesta, bowel segments were gently blotted dry and tissue weights recorded. The moisture content of cecal and colonic digesta was determined by freeze-drying a portion to constant weight. Digesta and fecal samples were diluted with a specified volume of internal standard (heptanoic acid) for analysis of short-chain fatty acid (SCFA) and mixed thoroughly for determination of pH using a standard glass electrode. The slurries were then stored frozen to await further analyses. For analysis of total and major individual SCFA, slurries were thawed, centrifuged, and concentrated by low-temperature vacuum microdistillation for quantification by glc.

1. Regina, A., Kosar-Hashemi, B., Li, Z., Pedler, A., Mukai, Y., Yamamoto, M., Gale, K., Sharp, P. J., Morell, M. K. & Rahman, S. (2006) Planta 222, 899-909.