Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (13): 2699-2709.doi: 10.3864/j.issn.0578-1752.2021.13.001

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

CRISPR/Cas9 Targeted Editing of OsIAA11 in Rice

LI ZhaoWei1(),LING DongLan1,SUN CongYing1,ZENG HuiLing1,LIU KaiJi1,LAN YingShan1,FAN Kai2,LIN WenXiong1()   

  1. 1College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002
    2College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2020-12-03 Revised:2021-01-21 Online:2021-07-01 Published:2021-07-12
  • Contact: WenXiong LIN E-mail:lizw197@163.com;wenxiong181@163.com

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Abstract:

【Objective】Auxin signaling pathway regulated by OsIAA11 plays an important role in the plant growth and response to various environment stresses, impacting on the final grain yield during the late stage. To explore the effect of OsIAA11 mutation on the yield, the OsIAA11 mutant plant was obtained using CRISPR/Cas9 system, and the agronomic characters of mutant plants were investigated in the field. 【Method】According to the principle of CRISPR/Cas9 technology, two 20 bp targeted sequences were designed in the first exon and second exon of the OsIAA11 genome sequence. The gene shuffling of non-specific target sites was eliminated using the blast analysis. The oligonucleotides of two target sites were inserted into the pYLgRNA-U6a and pYLgRNA-U6b plasmid vector, and then were amplified twice by PCR technology to construct U6a-IAA11-T1 and U6b-IAA11-T2 expression-boxes. The recombinant pYLCRISPR/Cas9-IAA11-T12 vector was obtained by linking two expression-boxes to pYLCRISPR/Cas9-MT vector. The pYLCRISPR/Cas9-IAA11-T12 vector was transformed into the callus ofjaponica rice ZH11 through Agrobacterium-mediated method. The T0 generation of transgenic plants was obtained by tissue culture, and the positive transgenic plants were selected through PCR method. The target sites of each T2 generation from T0 positive plants were determined via PCR and sequencing method, and finally the mutated genotypes were identified. Meanwhile, agronomic traits of the T2 generation were investigated in the field. 【Result】The pYLCRISPR/Cas9-IAA11-T12 expression vector was successfully transformed into the callus of ZH11. 25 transgenic lines were obtained, and 20 transgenic lines were identified as the positive plants. After analysis of T 2 transgenic plants via PCR and sequencing the target locus, 17 different homozygous mutations were identified in the two target sites of the OsIAA11 genomic sequence. Except for the single base insertion mutation of osiaa11-20-1, osiaa11-21-2, osiaa11-23, and osiaa11-25 in target 1 and osiaa11-22-2 in target 2, other genotypes mainly deleted small fragment base in target 1 and mutated a single base in target 2. Agronomic traits results showed that 17 osiaa11 mutants did not present a significant difference in plant height, panicle length, grain number per panicle, seed setting rate, 1000-grain weight, harvest index, and grain-straw ratio in comparison with the wild type. However, the osiaa11 displayed a significant decrease in effective spike rate, suggesting a more ineffective tiller phenotype. 【Conclusion】CRISPR/Cas9 technology successfully editedOsIAA11 and 17 different homozygous mutations genotypes were obtained. The mutant showed the reduced effective spike rate and increased ineffective tillers, indicating that OsIAA11 was related to the tiller bud appearance dominated by auxin.

Key words: rice, gene editing, OsIAA11, CRISPR/Cas9 technology, osiaa11 mutant

Table 1

The primer sequences used in this study"

引物名称Primer name 引物序列Primer sequence (5′-3′) 用途Usage
OsIAA11-T1-fwd gccgCGCGCGGCTTGTCGGGATC 靶序列1构建
Construction of target site 1
OsIAA11-T1-rev aaacGATCCCGACAAGCCGCGCG
OsIAA11-T2-fwd gttgAGCTACCCGGAGTTGTCCA 靶序列2构建
Construction of target site 2
OsIAA11-T2-rev aaacTGGACAACTCCGGGTAGCT
Hyp-F ACGGTGTCGTCCATCACAGTTTGCC T0代阳性转基因植株鉴定
Identification of the T0 positive transgenic plant
Hyp-R TTCCGGAAGTGCTTGACATTGGGGA
OsIAA11-T1-F AGACCATCGACCTCAAGC 靶点1测序检测
Sequencing for target 1
OsIAA11-T1-R GCTAACTAATGGCTCTTC
OsIAA11-T2-F GACGATGATGTCAACTTT 靶点2测序检测
Sequencing for target 2
OsIAA11-T2-R GATAATGGTAGAATAACTCA
Hyp-T2-F CTATTTCTTTGCCCTCGGAC T2代纯合突变植株体内潮霉素基因检测
Detection of the gene encoded for hygromycin in the T2 homozygous mutations
Hyp-T2-R GACGTCTGTCGAGAAGTTTCTG

Fig. 1

Target sites of the gRNA in the OsIAA11 gene locus, amino acid sequence structure of OsIAA11, and constructing style of the pYLCRISPR/Cas9-IAA11-T12 vector A: Position of two gRNA targets in the OsIAA11 gene locus; B: Amino acid sequence structure of OsIAA11; C: Cloning of two gRNA cassettes into the pYLCRISPR/Cas9-MT vector. LB: Left border; RB: Right border "

Fig. 2

Sequencing results for the two target sequences inserted in pYLCRISPR/Cas9-IAA11-T12 recombinant vector"

Fig. 3

Identification of the positive transgenic plants by PCR amplification -: Negative contro; +: Positive control; M: 2000 bp DNA marker"

Fig. 4

PCR identification and sequence alignment of osiaa11 mutants in comparison with the WT A: PCR amplification results of the DNA fragments near the targets edited of OsIAA11 for partial T2 generation ofosiaa11 mutants, the fragment length of target 1 and 2 is 443 bp and 469 bp, respectively, and WT is ZH11; B: sequence alignment of the edited targets for the osiaa11 mutants and WT line. The blue letters are the target genome sequence; The red capital letters denote PAM; Dashes strikethrough indicate the deleted bases; Insertion nucleotides are shown in red lowercase letters; -: Deletion; +: Insertion; WT: Wild type "

Fig. 5

PCR detection of the gene encoded for hygromycin in the T2 homozygous mutations -: Negative control; +: Positive control; M: 2000 bp DNA marker"

Table 2

Agronomic traits of different type osiaa11 mutants and its corresponding wild type grown in paddy field "

株系
Line 		株高
Plant height
(cm) 		有效穗数
Effective panicle
number per plant 		成穗率
Effective
spike rate (%) 		穗长
Length of main
panicle (cm) 		穗粒数
Grain number
per panicle 		结实率
Seed setting
rate (%) 		千粒重
1000-grain
weight (g) 		单株产量
Grain yield
per plant (g) 		收获指数
Harvest
index 		谷草比
Grain-straw
ratio
osiaa11-1-1 69.33±4.03abcd 6.00±0.82b 37.71±4.54b 19.93±0.93bc 128.17±24.12a 87.79±3.72ab 26.22±0.41a 11.21±2.04bcd 0.46±0.031ab 0.95±0.113ab
osiaa11-1-2 70.50±2.50abcd 7.00±0.00b 44.44±5.56b 20.35±0.62abc 120.50±9.84a 78.25±16.36ab 25.92±0.78a 12.26±1.24abcd 0.46±0.004ab 0.96±0.000ab
osiaa11-8-1 77.50±1.50a 10.50±2.50ab 44.07±4.07b 22.10±1.45abc 130.50±27.98a 86.08±6.46ab 25.98±0.98a 22.45±4.55a 0.44±0.020ab 0.88±0.073ab
osiaa11-8-2 74.00±0.82ab 9.67±1.70ab 48.73±4.37b 22.15±1.27abc 128.67±33.21a 88.57±6.58ab 25.68±0.53a 18.69±1.84abc 0.46±0.012ab 0.96±0.021ab
osiaa11-11-1 69.00±4.54bc 6.33±1.80b 43.27±7.73b 22.70±1.17a 113.67±23.50a 83.63±6.67ab 24.28±1.11a 10.51±2.67d 0.46±0.038ab 0.98±0.166ab
osiaa11-11-2 74.0±0.00abc 4.00±0.00b 26.67±0.00b 21.95±0.70abc 118.25±7.50a 88.65±2.29ab 23.32±0.78a 9.85±2.48d 0.41±0.000ab 0.76±0.000ab
osiaa11-17 67.17±1.34bcd 12.00±2.00a 40.53±4.64b 20.47±1.50bc 108.50±13.91a 90.27±8.36a 23.01±1.16a 18.30±3.14a 0.44±0.042ab 0.86±0.149ab
osiaa11-19-1 69.30±2.32bc 8.00±1.26b 43.87±3.32b 20.58±0.84abc 118.70±20.27a 83.94±5.15ab 23.21±0.84a 11.72±1.19bcd 0.43±0.051ab 0.84±0.182ab
osiaa11-19-2 66.33±1.89bcd 10.33±0.94ab 43.58±2.03b 21.15±0.80abc 109.50±11.38a 87.18±5.55ab 24.43±1.02a 12.20±0.27abcd 0.38±0.033ab 0.67±0.094ab
osiaa11-20-1 64.00±1.63cd 5.67±0.47b 50.56±7.49b 21.73±1.84abc 94.17±22.56a 80.43±13.87ab 24.42±1.49a 7.11±2.92d 0.43±0.067ab 0.90±0.269ab
osiaa11-20-2 65.50±1.50bcd 6.50±0.50b 54.89±8.74b 22.58±0.74abc 125.50±29.77a 84.28±7.52ab 24.68±0.02a 11.34±2.88bcd 0.52±0.059a 1.28±0.298a
osiaa11-21-1 60.50±1.50d 7.00±0.00b 46.88±3.12b 19.82±1.84bc 86.00±4.06a 80.67±15.64ab 21.85±3.35a 6.88±1.62d 0.44±0.017ab 0.85±0.054ab
osiaa11-21-2 61.00±0.00cd 4.00±0.00b 57.14±0.00b 18.50±0.40c 78.00±8.00a 64.70±0.41b 23.95±0.0a 3.16±0.00d 0.38±0.000ab 0.72±0.000ab
osiaa11-22-1 66.00±1.87cd 8.00±0.71ab 45.75±3.29b 21.09±1.27abc 101.12±14.10a 85.04±5.89ab 23.52±0.94a 11.05±1.67cd 0.44±0.017ab 0.85±0.054ab
osiaa11-22-2 65.25±1.09cd 6.50±0.50b 40.29±4.74b 20.50±0.79abc 103.25±19.27a 88.23±6.83ab 23.18±1.24a 9.10±0.43d 0.42±0.027ab 0.79±0.086ab
osiaa11-23 64.50±0.50cd 8.00±0.00ab 39.23±2.87b 21.08±0.89abc 124.50±10.62a 84.01±7.71ab 22.40±0.80a 12.29±0.19abcd 0.35±0.037ab 0.59±0.100b
osiaa11-25 72.50±0.50abc 11.00±1.00ab 38.52±1.48b 22.92±2.10ab 144.00±47.72a 85.26±7.35ab 24.52±1.08a 20.08±2.34ab 0.35±0.032b 0.58±0.081b
WT 69.60±0.80abc 8.00±2.00b 89.78±10.50a 21.16±1.29abc 113.70±16.80a 85.42±6.88ab 22.80±1.53a 10.65±2.75d 0.47±0.036ab 1.08±0.121ab
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