关键词: 玉米自交系京724, CRISPR/Cas9, 基因编辑, BADH2, 香味, 2-乙酰-1-吡咯啉

Abstract:

【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 161.82 μg·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.

Key words: maize elite inbred line JING724, CRISPR/Cas9, genome editing technology, BADH2, fragrance, 2-acetyl-1-pyrroline