大麦SS2a剪接位点单碱基突变导致淀粉发生明显变化

doi:10.1016/j.jia.2023.10.031

摘要/Abstract

摘要：

淀粉的生物合成是一个的复杂的过程依赖于多种酶的协调作用。抗性淀粉在小肠中不被消化，从而可以阻止了血糖指数的快速上升。淀粉合成酶2a（SS2a）是支链淀粉生物合成中的关键酶，对淀粉结构和性质有重要影响。本研究中，我们从大麦EMS突变体库中鉴定出了ss2a缺失突变体（M3-1413）。在突变体中，诱变产生的单碱基突变位于SS2a第一内含子的3'端的RNA剪接受体（AG），导致RNA不能正常剪辑，并产生两个异常ss2a转录本，导致ss2a基因失活。表型分析表明突变体M3-1413的淀粉结构和性质发生显著变化，具体为总淀粉含量降低，直链淀粉和抗性淀粉含量升高。本研究揭示了大麦ss2a突变机制及其对淀粉特性的影响，有助于推动大麦淀粉功能食品的开发应用。

Abstract:

Starch biosynthesis is a complex process that relies on the coordinated action of multiple enzymes. Resistant starch is not digested in the small intestine, thus preventing a rapid rise in the glycemic index. Starch synthase 2a (SS2a) is a key enzyme in amylopectin biosynthesis that has significant effects on starch structure and properties. In this study, we identified an ss2a null mutant (M3-1413) with a single base mutation from an ethyl methane sulfonate (EMS)-mutagenized population of barley. The mutation was located at the 3´ end of the first intron of the RNA splicing receptor (AG) site, and resulted in abnormal RNA splicing and two abnormal transcripts of ss2a, which caused the inactivation of the SS2a gene. The starch structure and properties were significantly altered in the mutant, with M3-1413 containing lower total starch and higher amylose and resistant starch levels. This study sheds light on the effect of barley ss2a null mutations on starch properties and will help to guide new applications of barley starch in the development of nutritious food products.

Key words: barley , EMS mutagenesis , starch synthase 2a , splicing site mutation , starch property , resistant starch

Bang Wang, Jing Liu, Xiaolei Chen, Qiang Xu, Yazhou Zhang, Huixue Dong, Huaping Tang, Pengfei Qi, Mei Deng, Jian Ma, Jirui Wang, Guoyue Chen, Yuming Wei, Youliang Zheng, Qiantao Jiang. 大麦SS2a剪接位点单碱基突变导致淀粉发生明显变化[J]. Journal of Integrative Agriculture, 2025, 24(4): 1359-1371.

Bang Wang, Jing Liu, Xiaolei Chen, Qiang Xu, Yazhou Zhang, Huixue Dong, Huaping Tang, Pengfei Qi, Mei Deng, Jian Ma, Jirui Wang, Guoyue Chen, Yuming Wei, Youliang Zheng, Qiantao Jiang. A barley SS2a single base mutation at the splicing site leads to obvious changes in starch[J]. Journal of Integrative Agriculture, 2025, 24(4): 1359-1371.

参考文献

Asare E K, Jaiswal S, Maley J, Baga M, Sammynaiken R, Rossnagel B G, Chibbar R N. 2011. Barley grain constituents, starch composition, and structure affect starch in vitro enzymatic hydrolysis. Journal of Agricultural and Food Chemistry, 59, 4743–4754.

Bertolini A C, Souza E J, Nelson J E, Huber K C. 2003. Composition and reactivity of A- and B-type starch granules of normal, partial waxy, and waxy wheat. Cereal Chemistry, 80, 544–549.

Cartegni L, Chew S L, Krainer A R. 2002. Listening to silence and understanding nonsense: Exonic mutations that affect splicing. Nature Reviews Genetics, 3, 285–298.

Chen L, Huang L, Min D, Phillips A L, Wang S, Madgwick P J, Parry M A J, Hu Y. 2012. Development and characterization of a new TILLING population of common bread wheat (Triticum aestivum L.). PLoS ONE, 7, 2930–2955.

Dhital S, Shrestha A K, Hasjim J, Gidley M J. 2011. Physicochemical and structural properties of maize and potato starches as a function of granule size. Journal of Agricultural and Food Chemistry, 59, 10151–10161.

Duque P. 2011. A role for SR proteins in plant stress responses. Plant Signaling & Behavior, 6, 49–54.

Englyst H N, Kingman S M, Cummings J H. 1992. Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46(Suppl 2), S33–S50.

Evers A D, O’brien L, Blakeney A B. 1999. Cereal structure and composition. Crop & Pasture Science, 50, 629–650.

Feng X, Rahman M M, Hu Q, Wang B, Karim H, Guzman C, Harwood W A, Xu Q, Zhang Y, Tang H, Jiang Y, Qi P, Deng M, Ma J, Lan J, Wang J, Chen G, Lan X, Wei Y, Zheng Y, et al. 2022. HvGBSSI mutation at the splicing receptor site affected RNA splicing and decreased amylose content in barley. Frontiers in Plant Science, 13, 1003333.

Graveley B R. 2000. Sorting out the complexity of SR protein functions. RNA, 6, 1197–1211.

Hazard B A, Zhang X, Colasuonno P, Uauy C, Beckles D M, Dubcovsky J. 2012. Induced mutations in the starch branching enzyme II (SBEII) genes increase amylose and resistant starch content in durum wheat. Crop Science, 52, 1754–1766.

Henikoff S, Till B J, Comai L. 2004. TILLING. Traditional mutagenesis meets functional genomics. Plant Physiology, 135, 630–636.

Jane J, Chen Y , Lee L, Mcpherson A E, Wong K, Radosavljević M, Kasemsuwan T. 1999. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chemistry, 76, 629–637.

Li J, Yeh A. 2001. Relationships between thermal, rheological characteristics and swelling power for various starches. Journal of Food Engineering, 50, 141–148.

Li W, Xiao X, Zhang W, Zheng J, Luo Q, Ouyang S, Zhang G. 2014. Compositional, morphological, structural and physicochemical properties of starches from seven naked barley cultivars grown in China. Food Research International, 58, 7–14.

Liu F, Romanova N, Lee E A, Ahmed R, Evans M, Gilbert E P, Morell M, Emes M J, Tetlow I J. 2012. Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules. The Biochemical Journal, 448, 373–387.

Lu X, Liu J, Ren W, Yang Q, Chai Z, Chen R, Wang L, Zhao J, Lang Z, Wang H, Fan Y, Zhao J, Zhang C. 2017. Gene-indexed mutations in maize. Molecular Plant, 11, 496–504.

Luo J, Ahmed R, Kosar-Hashemi B, Larroque O, Butardo V M, Tanner G J, Colgrave M L, Upadhyaya N M, Tetlow I J, Emes M J, Millar A A, Jobling S A, Morell M, Li Z. 2015. The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma. Theoretical and Applied Genetics, 128, 1407–1419.

Luo M, Ding J, Li Y, Tang H, Qi P, Ma J, Wang J, Chen G, Pu Z, Li W, Li Z, Harwood W A, Lan X, Deng M, Lu Z X, Wei Y, Zheng Y, Jiang Q. 2019. A single-base change at a splice site in Wx-A1 caused incorrect RNA splicing and gene inactivation in a wheat EMS mutant line. Theoretical and Applied Genetics, 132, 2097–2109.

Ma Z, Boye J I. 2018. Research advances on structural characterization of resistant starch and its structure-physiological function relationship: A review. Critical Reviews in Food Science and Nutrition, 58, 1059–1083.

Moreau C, Warren F J, Rayner T, Pérez-Moral N, Lawson D M, Wang T L, Domoney C. 2022. An allelic series of starch-branching enzyme mutants in pea (Pisum sativum L.) reveals complex relationships with seed starch phenotypes. Carbohydrate Polymers, 288, 119386.

Motilva M, Serra A, Borràs X, Romero M, Domínguez A, Labrador A, Peiró L. 2014. Adaptation of the standard enzymatic protocol (Megazyme method) to microplaque format for β-(1,3)(1,4)-d-glucan determination in cereal based samples with a wide range of β-glucan content. Journal of Cereal Science, 59, 224–227.

Murray M G, Thompson W F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8 19, 4321–4325.

Nakamura Y, Yuki K, Park S Y, Ohya T. 1989. Carbohydrate metabolism in the developing endosperm of rice grains. Plant and Cell Physiology, 30, 833–839.

Nilsson G, Gorton L, Bergquist K E, Nilsson U J. 1996. Determination of the degree of branching in normal and amylopectin type potato starch with 1H-NMR spectroscopy improved resolution and two-dimensional spectroscopy. Starch-Starke, 48, 352–357.

Nishi A, Nakamura Y, Tanaka N, Satoh H. 2001. Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm. Plant Physiology, 127, 459–472.

Raeker M O, Gaines C S, Finney P L, Donelson T. 1998. Granule size distribution and chemical composition of starches from 12 soft wheat cultivars. Cereal Chemistry, 75, 721–728.

Rahman S, Bird A R, Regina A, Li Z, Ral J, McMaugh S, Topping D L, Morell M. 2007. Resistant starch in cereals: Exploiting genetic engineering and genetic variation. Journal of Cereal Science, 46, 251–260.

Reddy A S N. 2001. Nuclear pre-mRNA splicing in plants. Critical Reviews in Plant Sciences, 20, 523–571.

Riaz A, Alqudah A M, Kanwal F, Pillen K, Ye L Z, Dai F, Zhang G P. 2022. Advances in studies on the physiological and molecular regulation of barley tillering. Journal of Integrative Agriculture, 21, 689–705.

Schmidt V, Kirschner K M. 2018. Alternative pre-mRNA splicing. Acta Physiologica, 222, e13053.

Schrader E K, Harstad K G, Matouschek A. 2009. Targeting proteins for degradation. Nature Chemical Biology, 5, 815–822.

Schultz J, Copley R R, Doerks T, Ponting C P, Bork P. 2000. SMART: A web-based tool for the study of genetically mobile domains. Nucleic Acids Research, 28, 231–234.

Serrat X, Esteban R, Guibourt N, Moysset L, Nogués S, Lalanne E. 2014. EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations. Plant Methods, 10, 5.

Sestili F, Janni M, Doherty A, Botticella E, D’Ovidio R, Masci S, Jones H D, Lafiandra D. 2010. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes. BMC Plant Biology, 10, 144.

Sestili F, Palombieri S, Botticella E, Mantovani P, Bovina R, Lafiandra D. 2015. TILLING mutants of durum wheat result in a high amylose phenotype and provide information on alternative splicing mechanisms. Plant Science, 233, 127–133.

South J B, Morrison W R. 1990. Isolation and analysis of starch from single kernels of wheat and barley. Journal of Cereal Science, 12, 43–51.

Umemoto T, Aoki N, Lin H, Nakamura Y, Inouchi N, Sato Y, Yano M, Hirabayashi H, Maruyama S. 2004. Natural variation in rice starch synthase IIa affects enzyme and starch properties. Functional Plant Biology, 31, 671–684.

Walsh S K, Lucey A, Walter J, Zannini E, Arendt E K. 2022. Resistant starch - An accessible fiber ingredient acceptable to the Western palate. Comprehensive Reviews in Food Science and Food Safety, 21, 2930–2955.

Yamamori M, Endo T. 1996. Variation of starch granule proteins and chromosome mapping of their coding genes in common wheat. Theoretical and Applied Genetics, 93, 275–281.

Yamamori M, Fujita S, Hayakawa K, Matsuki J, Yasui T. 2000. Genetic elimination of a starch granule protein, SGP-1, of wheat generates an altered starch with apparent high amylose. Theoretical and Applied Genetics, 101, 21–29.

Yan J, Jia J, Jiang L, Peng D, Liu S, Hou S, Yu J, Li H, Huang W. 2022. Resistance of barley varieties to Heterodera avenae in the Qinghai–Tibet Plateau, China. Journal of Integrative Agriculture, 21, 1401–1413.

Yang Q, Ding J, Feng X, Zhong X, Lan J, Tang H, Harwood W A, Li Z, Guzmán C, Xu Q, Zhang Y, Jiang Y, Qi P, Deng M, Ma J, Wang J, Chen G, Lan X, Wei Y, Zheng Y, et al. 2022. Editing of the starch synthase IIa gene led to transcriptomic and metabolomic changes and high amylose starch in barley. Carbohydrate Polymers, 285, 119238.

Zhang X, Colleoni C, Ratushna V, Sirghie-colleoni M, James M G, Myers A M. 2004. Molecular characterization demonstrates that the Zea mays gene sugary2 codes for the starch synthase isoform SSIIa. Plant Molecular Biology, 54, 865–879.

Zhang X, Gao X, Li Z, Xu L, Li Y, Zhang R, Xue J, Guo D. 2020. The effect of amylose on kernel phenotypic characteristics, starch-related gene expression and amylose inheritance in naturally mutated high-amylose maize. Journal of Integrative Agriculture, 19, 1554–1564.

Zobel H F. 1988. Starch crystal transformations and their industrial importance. Starch-Starke, 40, 1–7.