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Reduced expression of starch branching enzyme IIa and IIb in maize endosperm by RNAi constructs greatly increases the amylose content in kernel with nearly normal morphology

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Abstract

Main conclusion

RNAi technology was applied to suppress the expression of starch branching enzyme IIa and IIb and to increase amylose content in maize endosperm, and stably inherited high-amylose maize lines were obtained.

Amylose is an important material for industries and in the human diet. Maize varieties with endosperm amylose content (AC) of greater than 50 % are termed amylomaize, and possess high industrial application value. The high-amylose trait is controlled by multi-enzyme reaction and intricate gene–environment interaction. Starch branching enzymes are key factors for regulating the branching profiles of starches. In this paper, we report the successful application of RNAi technology for improving amylose content in maize endosperm through the suppression of the ZmSBEIIa and ZmSBEIIb genes by hairpin SBEIIRNAi constructs. These SBEIIRNAi transgenes led to the down-regulation of ZmSBEII expression and SBE activity to various degrees and altered the morphology of starch granules. Transgenic maize lines with AC of up to 55.89 % were produced, which avoided the significant decreases in starch content and grain yield that occur in high-amylose ae mutant. Novel maize lines with high AC offer potential benefits for high-amylose maize breeding. A comparison of gene silencing efficiency among transgenic lines containing different hpSBEIIRNA constructs demonstrated that (1) it was more efficient to use both ZmSBEIIa and ZmSBEIIb specific regions than to use the conserved domain as the inverted repeat arms; (2) the endosperm-specific promoter of the 27-kDa γ-zein provided more efficient inhibition than the CaMV 35S promoter; and (3) inclusion of the catalase intron in the hpSBEIIRNA constructs provided a better silencing effect than the chalcone synthase intron in the hpRNA construct design for suppression of the SBEII subfamily in endosperm.

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Abbreviations

AC:

Amylose content

ae:

Amylose-extender

AGPase:

ADP-glucose pyrophosphorylase

CTAB:

Cetyltrimethylammonium bromide

DP:

Degree of polymerization

DAP:

Days after pollination

GBSS:

Granule-bound starch synthetase

RNAi:

RNA interference

SBE:

Starch branching enzymes

DBE:

Starch debranching enzymes

SS:

Starch synthetases

WT:

Wild type

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Acknowledgments

We thank Liu, Q., Zhang, C., and Man, J. (Key Laboratories of Crop Genetics and Physiology of the Jiangsu Province and Plant Functional Genomics of the Ministry of Education, Yangzhou University) for assistance in GPC analysis. We thank Cong, H. (Life Science College of Shandong University) for help on performing SEM for observing the morphology of starch granules. We thank Li, S. (Life Science College of Shandong University) for the maize shoot-tip genetic transformation and cultivation. This work was supported by the Hi-Tech Research and Development (863 Program of China, 2012AA10A306).

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  1. School of Life Science, Shandong University, 27 Shanda South Road, Jinan, 250100, People’s Republic of China

    Yajie Zhao, Ning Li, Bei Li, Zhaoxia Li, Guangning Xie & Juren Zhang

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  1. Yajie Zhao
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  2. Ning Li
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Correspondence to Juren Zhang.

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Zhao, Y., Li, N., Li, B. et al. Reduced expression of starch branching enzyme IIa and IIb in maize endosperm by RNAi constructs greatly increases the amylose content in kernel with nearly normal morphology. Planta 241, 449–461 (2015). https://doi.org/10.1007/s00425-014-2192-1

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