High resolution melting analysis for the detection of EMS induced mutations in wheat SBEIIa genes.
High resolution melting analysis for the detection of EMS induced mutations in wheat SBEIIa genes.
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DOI：
10.1186/1471-2229-11-156
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发表时间：
2011-11-10
影响因子：
5.3
通讯作者：
Lafiandra D
中科院分区：
文献类型：
--
作者：
Botticella E;Sestili F;Hernandez-Lopez A;Phillips A;Lafiandra D
控制谷物淀粉中直链淀粉-支链淀粉的比例已被确定为生产具有新功能特性的淀粉的主要目标。据报道，在小麦中，通过转基因方法沉默淀粉分支酶基因可使直链淀粉含量增加高达总淀粉的70%，表现出新颖而有趣的营养特性。在这项工作中，淀粉分支酶IIa （SBEIIa）的功能被TILLING定位在面包小麦中。利用高分辨率聚合酶链反应（High Resolution Melting of PCR）对一个ems诱变的小麦群体进行了筛选，以鉴定六倍体基因组中编码目标酶的三个同源基因的功能单核苷酸多态性。结果表明，SBEIIa-A、SBEIIa-B和SBEIIa-D分别鉴定出56个、14个和53个新的等位基因变异。通过生物信息学方法评估突变对蛋白质结构和功能的影响。三个同源SBEIIa基因各鉴定出两个假定的无义或剪接位点突变的等位基因；qRT-PCR分析显示，它们的基因表达量显著降低，直链淀粉含量增加。对不同的单空同源物进行金字塔化，可以分离出双空突变体，其直链淀粉含量比对照增加了21%。TILLING已经成功地用于产生控制小麦直链淀粉含量的SBEIIa基因的新等位基因。单和双零SBEIIa基因型的直链淀粉含量显著增加。
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.
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影响因子：
5.3
作者：
Dong C;Vincent K;Sharp P
通讯作者：
Sharp P
影响因子：
5.1
作者：
Gady AL;Hermans FW;Van de Wal MH;van Loo EN;Visser RG;Bachem CW
通讯作者：
Bachem CW
影响因子：
11.6
作者：
JOFUKU, KD;SCHIPPER, RD;GOLDBERG, RB
通讯作者：
GOLDBERG, RB
影响因子：
4.8
作者：
Abad, MC;Binderup, K;Geiger, JH
通讯作者：
Geiger, JH
影响因子：
11.6
作者：
DEHESH, K;FRANCI, C;QUAIL, PH
通讯作者：
QUAIL, PH