Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (10): 2064-2072.doi: 10.3864/j.issn.0578-1752.2021.10.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Creation of New Maize Variety with Fragrant Rice Like Flavor by Editing BADH2-1 and BADH2-2 Using CRISPR/Cas9

ZHANG Xiang(),SHI YaXing(),LU BaiShan(),WU Ying,LIU Ya,WANG YuanDong(),YANG JinXiao(),ZHAO JiuRan()   

  1. Maize Research Center, Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing 100097
  • Received:2021-01-25 Accepted:2020-02-20 Online:2021-05-16 Published:2021-05-24
  • Contact: YuanDong WANG,JinXiao YANG,JiuRan ZHAO E-mail:zhangxiang@maizedna.org;shiyaxing@maizedna.org;lubaishan@maizedna.org;wangyuandong@maizedna.org;yangjinxiao@maizedna.org;maizezhao@maizedna.org

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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

Supplementary Fig.1

Diagram of the map of CRISPR/Cas9"

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5’-3’)		 目的
Purpose
Cas9-F1 TTGCAGTACGTAATGGACTACAAGGACCACGACG 构建骨架载体表达盒2
Construction of cassette2
Cas9-R1 AGTATCGGTCTCATGTCGAGTCCGATGGAATATTTCTTATCGG
Cas9-F2 CTTATCGGTCTCAGACATTGGCACGAATAGCGTCG
Cas9-R2 TTTCAGACTAGTCTATTTCTTCTTCTTAGCCTGTCC
t35s-F TATCGTACTAGTCTGAAATCACCAGTCTCTCTCTACAA
t35s-R CCACCAGGCGCGCCCCACTGGATTTTGGTTTTAGG
prZmUbi-F CGTCCGCCTAGGCTGCAGTGCAGCGTGAC 构建骨架载体表达盒3
Construction of cassette3
prZmUbi-R ATTTCATGGATCCCTGCAGAAGTAACACCAAACA
PMI-03-F ATTTCATGGATCCATGCAGAAGCTGATCAACA
tNos-R AGCAGCCCTGCAGGGATCTAGTAACATAGATGACACCGC
Target1-F TGCATCTCCGAAGAGAGCCTATTG
Target1-R AAACCAATAGGCTCTCTTCGGAGA
BH2-1-F GAGGAAACCTGAGCTGGCTAA ZmBADH2-1靶点突变检测
Mutation detection of ZmBADH2-1 target site
BH2-1-R ATGGTGATGAGGAAAGTTATCGA
BH2-2-F AGTGTGCAATTTTTTTTCCAGTTT ZmBADH2-2靶点突变检测
Mutation detection of ZmBADH2-2 target site
BH2-2-R GGCCCAACAAATACGTGAAA
PMI-F TTCGCCCTGACCCCCT T0代转基因阳性植株检测
T0 Transgenic plants detection
PMI-R TGGCAGGATATATTGTGGTGTAA
BH-2-1-F CTTGCCGATCCCTAGCGA ZmBADH2-2靶点突变检测
Mutation detection of ZmBADH2-2 target site
BH-2-1-R CGAGACCTAGCGCATCAG

Fig. 1

The phylogenetic tree of Oryza sativa, Zea mays and Arabidopsis thaliana BADH gene family Red words represented maize ZmBADH2-1 (Zm00001d050339) and ZmBADH2-2 (Zm00001d032257)"

Fig. 2

Diagram of the sgRNA design (A) and mutation analysis (B) on ZmBADH2-1 and ZmBADH2-2 The nucleotides with underline represented target sequence, the nucleotides with red font represented PAM sequence, the nucleotides with green font indicated bases of insertion, -: Base deletions, blue words indicated base substitutions"

Supplementary Fig.2

Gene-editing induced mutations caused abnormalities of ZmBADH2-1and ZmBADH2-2 protein sequences"

Supplementary Fig.3

T1 seeds of Zmbadh1/Zmbadh2 double mutants"

Fig. 3

Chromatography of 2-AP in Daohuaxiang seeds (A) and mass spectra of 2-AP in Daohuaxiang (B) and T1 seeds of BH02 line (C)"

Fig. 4

The chromatogram (A) and contents of 2-AP (B) in the seeds of gene-editing lines (n=3, mean±SD) Lower case letters represented significant difference (P<0.01) in 2-AP contents of different gene editing lines"

Supplementary Fig.4

Mutations and 2-AP content of Zmbadh2-2 mutants A: Diagram of the sgRNA design of ZmBADH2-2. Primers used to detect mutation were shown in supplementary table 1. B: Mutations of two gene editing line on ZmBADH2-2. C: 2-AP content of Nipponbare, Daohuaxiang, B104 maize inbred line, Zmbadh2-1/Zmnadh2-2 double mutant line BH02 and two gene editing line of Zmbadh2-2 mutant in B104 background. Lower case letters represented significant difference (P<0.01) in 2-AP contents of different gene editing lines"

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