利用CRISPR-Cas9技术编辑Badh2基因创制优质香型籼稻三系不育系  

Development of high-quality fragrant indica CMS line by editing Badh2 gene using CRISPR-Cas9 technology in rice(Oryza sativa L.)

作　　者：韦新宇 曾跃辉 杨旺兴 肖长春 候新坡 黄建鸿 邹文广 许旭明 WEI Xin-Yu;ZENG Yue-Hui;YANG Wang-Xing;XIAO Chang-Chun;HOU Xin-Po;HUANG Jian-Hong;ZOU Wen-Guang;and XU Xu-Ming(Biotechnology Research Institute,Sanming Academy of Agricultural Sciences,Sanming 365500,Fujian,China;Rice Research Institute,Sanming Academy of Agricultural Sciences,Sanming 365500,Fujian,China;Fujian Key Laboratory of Crop Genetic Improvement and Innovative Utilization for Mountain Area,Sanming 365500,Fujian,China)

机构地区：[1]三明市农业科学研究院生物技术研究所,福建三明365500 [2]三明市农业科学研究院水稻研究所,福建三明365500 [3]福建省(山区)作物遗传改良与创新利用重点实验室,福建三明365500

出　　处：《作物学报》2023年第8期2144-2159,共16页Acta Agronomica Sinica

基　　金：财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-01);福建省自然科学基金项目(2021J01535,2021J01536);三明市科技计划项目(2019-N-4)资助。

摘　　要：稻米香味是水稻的重要食味品质之一,其主要受第8染色体上编码甜菜碱脱氢酶基因Badh2控制,该基因突变可导致香味物质2-乙酰-1-吡咯啉(2-AP)的含量增加从而促进香味的产生。本研究以三明市农业科学研究院自主选育的优质籼型杂交稻保持系明太B为受体,利用CRISPR-Cas9技术对其Badh2基因进行编辑和敲除。获得2个T0代转基因纯合突变体植株并对其衍生的48个T1代单株进行鉴定和分析,获得1个不含转基因载体骨架且在第2外显子插入单个碱基T的纯合突变体株系明太B-badh2。利用半定量PCR和qRT-PCR技术以及气相色谱质谱联用仪(GC-MS)检测Badh2基因相对表达量和2-AP含量;同时采用农业行业标准(NY/T1433-2014)推荐的48对水稻SSR引物进行指纹图谱分析。结果表明,该株系Badh2基因RNA表达水平显著下调;籽粒中香味物质2-AP的含量显著增加;指纹图谱分析发现,仅1对引物Rm571在野生型和突变体之间鉴定到等位变异,两组材料遗传差异较小。此外,本研究还对野生型和突变体T2代植株表型性状、稻米蒸煮食味品质和外观品质指标进行了考察和测定分析。结果表明,所有指标在两组材料间均无显著差异。进一步采用测交和回交转育方法并结合Badh2位点测序分析,成功选育获得了其对应的纯合香型三系不育系明太A-badh2。通过与恢复系明恢703、明恢3009测配,其组合产量与国家审定品种明太优703、明太优3009相近且表现出较强的超标优势。此外,通过与香型恢复系明恢1831测配后发现其组合籽粒中香味物质2-AP含量极显著高于对照组合明太A/明恢1831。因此,利用CRISPR-Cas9基因编辑技术,可对水稻香味基因Badh2进行精准定向编辑和敲除,实现对水稻香味性状的改良,为创制香型籼稻不育系提供理论指导,从而加快香型杂交稻育种进程。Fragrance is one of the important traits for quality improvement in rice,which is mainly controlled by the Badh2 gene encoding a betaine aldehyde dehydrogenase on chromosome 8.The mutation of Badh2 gene can increase the content of 2-acetyl-1-pyrroline(2-AP)and promote the fragrance production in rice.In this study,the Badh2 gene in Mingtai B(MTB),an elite maintainer line of Indica hybrid rice showing high eating quality bred by Sanming Academy of Agricultural Sciences,was edited and knocked out by using CRISPR-Cas9 technology.Two T0 transgenic lines carrying homozygous mutation on the loci of Badh2 were generated,and 48 T1 individuals derived from these two plants were genotyped by PCR amplification and sequencing analysis.A mutant line named MTB-badh2,which had a single T nucleotide insertion in the second exon of Badh2 without the vector skeleton,was finally obtained.In our study,the expression of Badh2 and the content of 2-AP were determined by semi-quantitative RT-PCR,qRT-PCR,and gas chromatography-mass spectrometry(GC-MS),respectively.Simultaneously,the 48 pairs of SSR markers recommended by“Sector Standard of Agriculture(NY/T 1433-2014)”were used to further analyze the DNA fingerprint and genetic diversity of the MTB and the MTB-badh2.The results showed that the Badh2 RNA level was significantly decreased in the MTB-badh2 compared with the wild-type MTB.In addition,the 2-AP content was dramatically increased in MTB-badh2.DNA fingerprint analysis revealed that only the Rm571 primer pair was specific for identifying allelic variation between wild type and MTB-badh2,suggesting that the genetic diversity between wild type and MTB-badh2 was very low.Furthermore,agronomic phenotype,cooking and eating quality,appearance quality of wild type,and MTB-badh2 were investigated and analyzed.There was no significant difference between MTB and MTB-badh2.The corresponding stable fragrant CMS line Mingtai A-badh2(MTA-badh2)carrying homozygous mutant on the locus of Badh2 was successfully generated by the conventional te

关 键 词：水稻 CRISPR-Cas9 基因编辑 香味 Badh2 2-乙酰-1-吡咯啉(2-AP) 

分 类 号：S511.21[农业科学—作物学]