Abstract：
Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α\'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.
摘要：
大豆β-伴大豆球蛋白是一种主要的过敏原，对大豆的营养特性产生不利影响。大豆缺乏β-伴大豆球蛋白与低变应原性和高营养价值有关。长基因间非编码RNA（lincRNAs）调节基因表达，被认为是基本生物过程的重要调节因子。尽管越来越了解lincRNAs的功能，关于lincRNAs对大豆β-伴大豆球蛋白积累的影响知之甚少。当前的研究提出了对定位到β-伴大豆球蛋白α亚基基因座的基因间非编码区的lincRNAlincCG1的鉴定。克隆了全长lincCG1序列，发现该序列可通过顺式和反式调节机制调节大豆种子贮藏蛋白（SSP）基因的表达。使用成簇的间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）系统产生的功能丧失lincCG1突变导致致敏性α'-，α-,和大豆β-伴大豆球蛋白的β-亚基以及较高的蛋白质含量，含硫氨基酸,和游离的精氨酸.显性无效等位基因LincCG1，因此，与lincCG1基因编辑品系相关的β-伴大豆球蛋白缺陷表型以孟德尔方式被后代稳定遗传。因此，显性无效等位基因LincCG1可用于工程化/开发新的低变应原性大豆品种。此外，在T1代中获得了无Cas9和β-伴大豆球蛋白缺陷的纯合突变体系。这项研究是首次使用CRISPR/Cas9技术编辑与大豆变应原蛋白β-伴大豆球蛋白相关的lincRNA基因。此外，这项研究揭示了lincCG1在调节β-伴大豆球蛋白亚基基因簇的表达中起着至关重要的作用，除了强调使用CRISPR/Cas9系统调节lincRNAs的效率外，从而调节大豆种子成分。
