摘要
【目的】香味是作物的重要食味品质之一。2-乙酰-1-吡咯啉(2-acetyl-1-pyrroline,2-AP)是主要香味物。BADH2是控制水稻等作物香味性状的关键基因,敲除该基因可以产生香味稻米。利用CRISPR/Cas9基因编辑技术在北京市农林科学院自育的玉米骨干亲本京724上敲除BADH2同源基因,以期获得有香米味道的玉米新种质材料。【方法】利用Ensembl数据库在线BLAST工具,将水稻OsBADH2蛋白序列在拟南芥、水稻和玉米蛋白序列数据库中进行序列比对,获得上述3个物种的BADH基因家族成员,并利用UniProt蛋白数据库中的结构域信息进行验证。进一步使用MEGA软件进行系统进化分析,获得玉米BADH2同源基因作为候选编辑靶标。基于CRISPR/Cas9基因编辑技术的原理,在候选基因的外显子处设计特异性靶点,并构建入CRISPR/Cas9基因编辑载体。再以玉米自交系京724为受体,利用农杆菌介导的遗传转化方法,通过磷酸甘露糖异构酶基因(phosphomannose isomerase,PMI)抗性筛选获得阳性转基因植株。转基因株系经测序明确其在靶基因中产生的突变类型。利用气相色谱质谱联用仪(gas chromatography-mass spectrometry,GC-MS)检测基因编辑株系T1籽粒中香米主要香味物质2-AP的含量,以确认京724在基因编辑前后2-AP含量的变化。【结果】系统进化分析发现,玉米中存在2个BADH2同源基因,分别命名为ZmBADH2-1和ZmBADH2-2。ZmBADH2-1位于第4染色体,ZmBADH2-2位于第1染色体。2个基因均包含15个外显子和14个内含子,第4外显子间的核苷酸序列高度同源。在2个基因的第4外显子区域设计靶点并构建入CRISPR/Cas9基因编辑载体,通过遗传转化获得28株转基因株系。PCR扩增及测序分析结果显示,其中10株材料的2个ZmBADH2s在靶点区域均发生突变,突变基因型包括双等位突变和多等位突变,突变类型为不同数量的碱基缺失和插入。质谱检测结果显示玉米ZmBADH2双基因突变体籽粒中存在与香稻同样成分的2-AP。随机选取的4个T_(1)代基因编辑株系籽粒中,2-AP平均含量分别为438.29、404.63、348.65和161.82μg·kg-1,而未经过编辑的京724中未检测到2-AP。【结论】利用CRISPR/Cas9技术对玉米ZmBADH2-1和ZmBADH2-2同时进行定点敲除,创制出籽粒中具有香米味道的玉米骨干亲本新种质材料。
【Objective】Fragrance is an important trait for quality improvement of crops.The 2-acetyl-1-pyrroline(2-AP)is the major component of the aroma flavor.BADH2 controls fragrance in plants,and its null or weak alleles lead to 2-AP accumulation.In this study,the fragrance related genes were modified in JING724,a maize elite inbred line invented by Beijing Academy of Agriculture and Forestry Sciences,using CRISPR/Cas9 to improve its trait of fragrance【Method】To find BADH gene family of target species,OsBADH2 protein sequence was used to search against protein databases of Arabidopsis,rice and maize with the Ensembl online BLAST tool.All BADH family members were verified by protein domain information in UniProt database.Furthermore,phylogenetic analysis conducted in MEGA software was used to search for maize BADH2 homologs as gene-editing targets.Based on principles of CRISPR/Cas9,we designed highly specific sgRNA to target the candidate genes.The CRISPR/Cas9 vector containing this sgRNA was introduced into the maize variety JING724 by Agrobacterium-mediated transformation.We obtained transgenic maize plants with PMI resistance.Sanger sequencing was used to confirm the CRISPR/Cas9-mediated mutations.Finally,we used gas chromatography-mass spectrometry(GC-MS)to measure the 2-AP content in T1 seeds of the gene-editing lines.【Result】Phylogenetic analysis showed that there were two BADH2 homologs in the maize gnome,subsequently they were named ZmBADH2-1 and ZmBADH2-2.ZmBADH2-1 is located in chromosome 4,whereas ZmBADH2-2 was in chromosome 1.Both genes have 15 exons and 14 introns.The 4th exon of ZmBADH2-1 shares high nucleotide identity with that of ZmBADH2-2.A specific target sequence,which is located in the 4th exons of both genes,was designed and introduced into a CRISPR/Cas9 vector.Using this vector,10 gene-editing lines were acquired after maize transformation.PCR amplification and sanger sequencing revealed that,in each of the 10 gene-editing lines,different type of insertions or deletions were introduced into the target sites of both ZmBADH2 genes successfully.Genotypes of mutations included biallelic and multi-allelic mutations.Mess spectra analysis showed that Zmbadh2-1/Zmbadh2-2 double mutants had 2-AP,which is the same substance of flavor with that in fragrant rice.Using GC-MS,we found that 2-AP contents in grains gathered from four randomly selected T0 gene-editing lines were 438.29,404.63,348.65 and 161.82μg·kg^(-1),respectively.On the contrary,no 2-AP was detected in JING724 wild type.【Conclusion】With site specific mutations introduced into ZmBADH2-1 and ZmBADH2-2 simultaneously using CRISPR/Cas9,new maize variety with fragrant rice like flavor was created successfully.
作者
张翔
史亚兴
卢柏山
武莹
刘亚
王元东
杨进孝
赵久然
ZHANG Xiang;SHI YaXing;LU BaiShan;WU Ying;LIU Ya;WANG YuanDong;YANG JinXiao;ZHAO JiuRan(Maize Research Center,Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding,Beijing 100097)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第10期2064-2072,I0002,I0003,I0004,共12页
Scientia Agricultura Sinica
基金
北京学者(BSP041)。