Field assessment of ultra-low asparagine, low acrylamide, gene edited wheat

大众食品中丙烯酰胺的存在已经成为食品工业及其供应链面临的最困难的问题之一。丙烯酰胺是一种污染物，由游离（非蛋白质）形式的天冬酰胺氨基酸和葡萄糖、果糖和麦芽糖等糖形成。该反应发生在油炸、烘烤、烘烤、烘烤和高温加工过程中。丙烯酰胺被列为可能致癌物质，高剂量会影响发育和生育。小麦和其他谷物制成的产品是膳食丙烯酰胺的主要来源。法规（EU） 2017/2158（2018）涵盖了食品中的丙烯酰胺，该法规为食品中丙烯酰胺的存在设定了基准水平，描述了强制性缓解措施，并要求所有食品企业监测其产品中丙烯酰胺的含量。用于限制加工过程中丙烯酰胺形成的方法并不适用于所有产品，可能会对产品质量产生不利影响，而且实施起来成本高昂。我们的方法是降低作物产品形成丙烯酰胺的可能性：对于小麦和其他谷物，这意味着降低谷物中游离天冬酰胺的浓度。该项目源于之前由bbsrc资助的工作，我们已经证明了小麦籽粒中游离天冬酰胺浓度的巨大品种差异，并表明它对缺硫和其他营养和环境压力因素以及疾病有高度反应。天冬酰胺是由一种叫做天冬酰胺合成酶的酶合成的，小麦有五个编码这种酶的基因。其中一个基因，TaASN2，已经成为一个遗传靶标，因为它在谷物中高度活跃。在我们目前的工作中，洛桑研究所（Rothamsted Research）、布里斯托尔大学（university of Bristol）以及合作伙伴AHDB、KWS、Limagrain、RAGT、Saaten Union和先正达（Syngenta）的团队使用了基因组编辑技术CRISPR-Cas9来敲除TaASN2基因。此外，我们还筛选了一个通过化学诱变引入突变的小麦群体（自20世纪50年代以来一直用于植物育种的一种更古老的技术），并确定了小麦基因组中6种TaASN2版本中每一种都有突变的品系。我们的一个合作伙伴正在对这些品系中的TaASN2突变进行“堆叠”，部分基因敲除将在项目的第2年实现。其中一个CRISPR品系中游离天冬酰胺的浓度降低了大约90%。该项目的目的是继续与布里斯托尔大学、AHDB、利马格兰、RAGT、Saaten Union和先正达的合作，对这些超低天冬酰胺小麦品系进行田间试验，以期为英国市场开发低丙烯酰胺小麦品种。在第一年，我们将为试验囤积种子。我们还将调查CRISPR系的发芽率：迄今为止，它们的发芽率很低，但这可以通过低浓度的天冬酰胺处理来逆转。我们将检查天冬酰胺和其他潜在治疗方法的有效性和实用性。我们还将开发遗传标记/工具，使育种者能够将超低天冬酰胺性状整合到育种品系中。最后，我们将准备一份风险评估，并提交一份允许进行实地试验的申请。在第二年，我们将进行田间试验，分析低天冬酰胺品系的出苗率、物理特性、发育差异、组成、产量和其他农艺特性。我们还将进行温室试验，以确定低天冬酰胺品系对缺硫的反应是否与普通小麦相同。该项目将成为作物基因编辑的一个里程碑，据我们所知，这是英国和欧洲首次进行基因编辑小麦的田间试验，而如果这些作物在田间试验中表现良好，AHDB和小麦育种家的参与将为商业化提供一条直接途径。

The presence of acrylamide in popular foods has become one of the most difficult problems facing the food industry and its supply chain. Acrylamide is a contaminant that forms from an amino acid called asparagine in its free (non-protein) form and sugars such as glucose, fructose and maltose. The reaction occurs during frying, baking, roasting, toasting and high-temperature processing. Acrylamide is classified as probably cancer-causing in humans and affects development and fertility at high doses. Products made from wheat and other cereals are major sources of dietary acrylamide.Acrylamide in food is covered by regulation (EU) 2017/2158 (2018), which set Benchmark Levels for the presence of acrylamide in food, described compulsory mitigation measures and required all food businesses to monitor the levels of acrylamide in their products. Methods developed to limit acrylamide formation during processing do not work for all products, may adversely affect product quality and are expensive to implement. Our approach has been to lower the acrylamide-forming potential of crop products: for wheat and other cereals that means reducing the concentration of free asparagine in the grain. The project arises from previous BBSRC-funded work in which we have demonstrated large varietal differences in free asparagine concentration in wheat grain and shown it to be highly responsive to sulphur deficiency and other nutritional and environmental stress factors, as well as disease. Asparagine is made by an enzyme called asparagine synthetase and wheat has five genes that encode this enzyme. One of these genes, TaASN2, has emerged as a genetic target because it is highly active specifically in the grain. In our current work, involving teams at Rothamsted Research, Univ. of Bristol and partners AHDB, KWS, Limagrain, RAGT, Saaten Union and Syngenta, we have used the genome editing technique, CRISPR-Cas9, to knock out TaASN2 genes. In addition, we have screened a wheat population in which mutations have been introduced by chemical mutagenesis (a much older technique that has been used in plant breeding since the 1950s) and identified lines with mutations in each of the six versions of TaASN2 in the wheat genome. The TaASN2 mutations in these lines are being 'stacked' by one of our partners and partial knockouts will be available by Year 2 of the project. The concentration of free asparagine in one of the CRISPR lines is reduced by approximately 90%. The aim of this project, which will continue the partnership with Univ. of Bristol, AHDB, Limagrain, RAGT, Saaten Union and Syngenta, is to undertake field trials of these ultra-low asparagine wheat lines with a view to the development of low acrylamide wheat varieties for the UK market. In year 1 we will bulk up seed for the trial. We will also investigate the germination rate of the CRISPR lines: they have shown poor germination so far but this can be reversed by treatment with low concentrations of asparagine. We will check the efficacy and practicality of the asparagine and other potential treatments. We will also develop genetic markers/tools to enable breeders to integrate the ultra-low asparagine trait into breeding lines. Lastly, we will prepare a risk assessment and submit an application for permission to hold the field trial. In year 2 we will conduct the field trial, analysing the low asparagine lines for emergence, physical characteristics, developmental differences, composition, yield and other agronomic characteristics. We will also conduct glasshouse experiments to ascertain whether the low asparagine lines respond to sulphur deficiency in the same way as normal wheat. The project would represent a landmark for crop gene editing, with the field trial, to our knowledge, being the first for gene edited wheat in the UK and Europe, while the involvement of AHDB and wheat breeders will provide a direct pathway to commercialisation if the plants perform well in the field trial.