Department of Agriculture, Forests, Nature and Energy, University of Tuscia, 01100 Viterbo, Italy.
BMC Plant Biol. 2011 Nov 10;11:156. doi: 10.1186/1471-2229-11-156.
Manipulation of the amylose-amylopectin ratio in cereal starch has been identified as a major target for the production of starches with novel functional properties. In wheat, silencing of starch branching enzyme genes by a transgenic approach reportedly caused an increase of amylose content up to 70% of total starch, exhibiting novel and interesting nutritional characteristics. In this work, the functionality of starch branching enzyme IIa (SBEIIa) has been targeted in bread wheat by TILLING. An EMS-mutagenised wheat population has been screened using High Resolution Melting of PCR products to identify functional SNPs in the three homoeologous genes encoding the target enzyme in the hexaploid genome.
This analysis resulted in the identification of 56, 14 and 53 new allelic variants respectively for SBEIIa-A, SBEIIa-B and SBEIIa-D. The effects of the mutations on protein structure and functionality were evaluated by a bioinformatic approach. Two putative null alleles containing non-sense or splice site mutations were identified for each of the three homoeologous SBEIIa genes; qRT-PCR analysis showed a significant decrease of their gene expression and resulted in increased amylose content. Pyramiding of different single null homoeologous allowed to isolate double null mutants showing an increase of amylose content up to 21% compared to the control.
TILLING has successfully been used to generate novel alleles for SBEIIa genes known to control amylose content in wheat. Single and double null SBEIIa genotypes have been found to show a significant increase in amylose content.
改变谷物淀粉中直链淀粉-支链淀粉比例已成为生产具有新型功能特性淀粉的主要目标。在小麦中，通过转基因方法沉默淀粉分支酶基因，导致直链淀粉含量增加到总淀粉的 70%，表现出新颖有趣的营养特性。在这项工作中，通过 TILLING 技术靶向面包小麦中的淀粉分支酶 IIa（SBEIIa）。使用高分辨率熔解曲线 PCR 产物筛选 EMS 诱变的小麦群体，以鉴定编码六倍体基因组中靶酶的三个同源基因中的功能 SNP。
这项分析分别在 SBEIIa-A、SBEIIa-B 和 SBEIIa-D 三个同源基因中鉴定出了 56、14 和 53 个新的等位基因变异。突变对蛋白质结构和功能的影响通过生物信息学方法进行了评估。在三个同源 SBEIIa 基因中，每个基因都鉴定出了两个可能的无效等位基因，它们包含无义或剪接位点突变；qRT-PCR 分析表明它们的基因表达显著下降，导致直链淀粉含量增加。不同单 null 同源基因的级联可以分离出双 null 突变体，与对照相比，直链淀粉含量增加了 21%。
TILLING 已成功用于生成已知控制小麦直链淀粉含量的 SBEIIa 基因的新型等位基因。单和双 null SBEIIa 基因型表现出直链淀粉含量的显著增加。
