Plant Biotechnology Journal, Journal Year: 2020, Volume and Issue: 19(1), P. 11 - 13

Published: June 17, 2020

The genetic diversity and phenotypic variability of crop agronomic traits is valued by breeders for their benefits in breeding but are limited most target traits. Genome editing has proved to be a powerful tool quick efficient creation continuous beneficial variation (Eshed Lippman, 2019). rice Waxy (Wx) gene (LOC_Os06g04200) encodes granule-bound starch synthase I (GBSSI), which determines the amylose content (AC) endosperm controlling synthesis. This one major contributors eating cooking quality (ECQ) (Li et al., 2016), an attribute that receiving increased attention society because improvement people's living standards. Rice AC ranges from 0 ~30% depending on presence different Wx alleles, with Wxa(relatively high more than 20%) Wxb (intermediate 14 ~18%) being alleles found indica japonica varieties, respectively (Teng 2012). Amino acid changes Wx/GBSSI protein can affect grain, as well-known 'soft rice' varieties (AC 7%–10%) genotypes Wxop/hp, WxmqorWxmp (Zhu 2015), all have non-synonymous mutations N-terminal domain (Momma Fujimoto, As moderately low (<12%), become popular commercially Gilbert, 2018), both traditional molecular approaches including CRISPR/Cas9-mediated knockout (Ma 2015; Zhang 2018) been used mutate reduce grain. However, only number mutants generated, far fewer needed meet diverse demands ECQ. We hypothesized grain could fine-turned generating series novel amino substitution(s) close allele responsible sites (such residues 158th inWxmq or Wxmp, 191th 165th Wxop/hp allele) state-of-the-art base editing. Based requirements cytidine editors (CBEs) (Zong 2017), we designed three sgRNAs targeting third (target site1, TS1), fourth site 2, TS2) fifth site3, TS3) exons (Figure 1a), were mentioned sites. cloned into vector pH-nCas9-PBE generate vectors PBE-TS1, PBE-TS2 PBE-TS3, respectively. resulting plasmids individually introduced cultivar Nipponbare (NIP) Agrobacterium-mediated transformation. A total 5, 10 7 independent T0 transgenic lines, respectively, 5 2 representative edited lines 1b) taken T1 generation; T-DNA-free homozygous individuals then chosen analysed detail. observed variety mutation types position substitutions within window; these reflected present parental suggesting T0alleles faithfully transmitted next generation 1b). Using TS1, line, Wxm5 (from line B7-2/6), carrying C2, 3, 5-to-T2, transition led P124F R125W was obtained; using TS2, four Wxm6 B6-29, G6, 7-to-A6, leading G159K mutation), Wxm7 B2-25, G6-to-C6 transversion G159A Wxm8 G1-to-A1 transversion, D161N mutations) Wxm9 B1-68, G4-to-T4 G6-to-A6 transition, G159E V160F identified; TS3, two Wxm10 B2-21, C5, 6-to-T5, 6 T178I mutation) Wxm11 C5-to-G5 C6-to-T6 T178S obtained 1c). In addition, seven (Wxm5-Wxm11), failed find any potential off-target 1d). To determine effect AC, measured apparent contents (AACs) grains mutant NIP (Wxb) control Nangeng9108 (NG9108) (Wxmp) 1e). Notably, had AAC (1.4 ± 0.2%) glutinous rice. AACs (11.9 0.1%), (11.3 (9.8 (7.9 0.1%) significantly lower (14.4 0.2%), comparable NG9108 (9.6 0.2%). (5.8 (4.2 lay between those Wxm5. GBSSI activities developing seeds Wx-edited days after flowering ranged 231.5 16.5 712.1 54.1 nmol/g/min 1f), NIP. reduced likely due amount 1g). These results demonstrate TS1-TS3 indeed abundance activity decrease seeds. general, appearance milled (especially transparency grain) negatively correlated 2018). 7%–10% (e.g. NG9108) semi-translucent while < 2% opaque. compared (10% moisture) (T2 generation) NG9108. indicated Figure 1h, opaque rice-like, consistent AAC. Wxm8, like Interestingly, Wxm6, andWxm10, 9.8%–11.9%, tended rather NG9108, almost transparent semi-translucent, indicating successfully generated germ plasms (~10%) without affecting achieved confirmed other Jingeng818 (JG818) Suijing18 (SJ18), NIP, exampleWxm5, 1i), strategy this study reliable fine-tune elite varieties. summary, base-editing system create 1.4%–11.9% goal over range 0%–12% enrich materials available breeders. Furthermore, speculated C-terminal domain) and/or Wxa) further extend AC. shows it possible obtain substituting many individual acids critical domains genes economically important provides new breeding. work supported grants National Transgenic Science Technology Program (2019ZX08010-003), Natural Foundation China (31701511), Jiangsu Province (BK20170610), Key R&D (2018YFA0900600) State Laboratory Plant Cell Chromosome Engineering (PCCE-KF-2020-01). authors submitted patent application based reported paper. J. Y., C. G. Y. X. research; X., Q. L., F. W., Z. C., W. F., T., J., R. performed B. contributed writing; Q-H. wrote manuscript.

Language: Английский