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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (6): 844-851.doi: 10.3724/SP.J.1006.2018.00844

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

Construction of tms5 Mutants in Rice Based on CRISPR/Cas9 Technology

Zhong-Ming HUANG1,Yan-Biao ZHOU2,3,*(),Xiao-Dan TANG3,Xin-Hui ZHAO3,Zai-Wei ZHOU3,Xing-Xue FU3,Kai WANG4,Jiang-Wei SHI3,Yan-Feng LI4,Chen-Jian FU2,3,Yuan-Zhu YANG1,2,3,4,*()   

  1. 1 College of Agronomy, Hunan Agricultural University, Changsha 410128, Hunan, China
    2 Yuan Longping High-Tech Agriculture Co. Ltd., Hunan / Hunan Engineering Laboratory for Disease and Pest Resistance Rice Breeding, Changsha, 410001, Hunan, China
    3 Hunan Longping High-Tech Seeds Science Academy Co. Ltd., Changsha 410119, Hunan, China
    4 College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China
  • Received:2017-10-25 Accepted:2018-03-26 Online:2018-06-12 Published:2018-04-08
  • Contact: Yan-Biao ZHOU,Yuan-Zhu YANG E-mail:zhouyb@lpht.com.cn;yzhuyah@163.com
  • Supported by:
    This study was supported by the Major Project of China on New Varieties of GMO Cultivation(2016ZX08001-004);the Key Research Program of Hunan Province(2016JC2026)

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Abstract:

In order to create excellent temperature sensitive sterile line and promote the development of two line hybrid rice breeding. a 20 bp guide RNA (gRNA) was targeted to the first exon of TMS5, a thermo-sensitive genic male sterile gene as the editable object in present study, and driven by the rice U3 promoter. The target site of gRNA was ligated into the vector pCAMBIA1301, and its transgenic lines were obtained via Agrobacterium-mediated transformation. The genomic DNA of T0 transgenic lines was then extracted, and the TMS5 locus was detected by PCR and sequenced. The mutagenesis frequency for TMS5 was 63.89%, of which the frequency of homozygous deletion mutation was 34.78%. Furthermore, the fertility transition temperature and agronomic traits of T1 homozygous deletion mutants were investigated, showing that the fertility transition temperature of tms5 mutants was 28°C. Both seed setting rate and grain yields in tms5 mutants were significantly decreased compared with those in wild-type plants in paddy field condition. Together, the successful construction of tms5 mutants lays a material foundation for breeding the thermo-sensitive male sterile line of japonica rice.

Key words: rice, genome editing, CRISPR/Cas9, TMS5, thermo-sensitive genic male sterile

Fig. 1

Schematic diagram of the targeted site in TMS5 The red letters are the target genome sequences; The blue letters are the protospacer adjacent motif (PAM) sequences."

Table 1

Primer sequences used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
Target-1F TGGCGAACAGCGGCAAGTCATCGC
Target -1R AAACGCGATGACTTGCCGCTGTTC
M13F GTAAAACGACGGCCAG
GUS-JC-F CGTCCGTCCTGTAGAAACCC
GUS-JC-R GTGCGGATTCACCACTTGC
TMS5-CX-F TCCAACGCATAGCAGTAGTCG
TMS5-CX-R TGCCATCGTATCTCCGGTAAA

Fig. 2

Schematic diagram of the CRISPR/Cas9-TMS5 vector construction A: schematic diagram of the recombinant vector of 18T-Cas9-TMS5-gRNA; B: T-DNA fragment of pCAMBIA1301 vector; C: schematic diagram of the pCAMBIA1301-Cas9-TMS5-gRNA construct. LB: left border; RB: right border."

Fig. 3

Identification of the pCAMBIA1301-Cas9-TMS5-gRNA plasmid by restriction enzyme digestion and PCR A: identification of the pCAMBIA1301-Cas9-TMS5-gRNA plasmid digested with Kpn I and Hind III; B: identification of the pCAMBIA1301-Cas9-TMS5-gRNA plasmid in Agrobacterium tumefaciens by PCR with primers of GUS-JC-F and GUS-JC-R. M: DL2000; 1: 18T-Cas9-TMS5-gRNA; 2: pCAMBIA1301; 3: pCAMBIA1301-Cas9-TMS5-gRNA."

Supplementary Fig. 1

Analysis of mutation sequences of target The red letters are the target genome sequences; the blue letters are PAM; the green letters are the insert base; +: Insertion; WT: Wild-type; Ho: homozygous mutation; He: heterozygous mutation."

Fig. 4

Identification of the transgenic plants A: identification of the positive transgenic plants by PCR; B: identification of the positive transgenic plants by GUS histochemical staining. M: DL2000; +: positive control; 1-18: transgenic plants."

Fig. 5

Mutation types and frequencies at the TMS5 loci in T0 transgenic plants A: Sequence alignment of homozygous mutation types; B: Frequency of different base inserts in homozygous mutation types. The red letters are the target genome sequences; The blue letters are PAM; The green letters are the insert base."

Fig. 6

Pollen fertility and seed setting rate of wild-type (WT) and tms5 mutant at different temperatures treatments Data are presented as average values ± standard error of the mean. **, significant difference between tms5 mutant and WT (P < 0.01)."

Table 2

Agronomic traits of the wild-type (WT) and tms5 mutant grown in paddy ?eld"

株系
Line		 株高
Plant height
(cm)		 千粒重
1000-grain weight
(g)		 有效穗
Panicle number
per plant		 结实率
Seed setting rate
(%)		 单株产量
Grain yield per plant
(g)
WT 78.96±3.56 20.82±0.97 9.3±1.87 72.21±3.57 17.30±1.09
tms5 78.57±3.87 21.20±0.88 9.85±1.79 61.60±3.43** 15.22±1.05**
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