CRISPR/Cas9-mediated restoration of Tamyb10 to create pre-harvest sprouting-resistant red wheat.
CRISPR/Cas9-mediated restoration of Tamyb10 to create pre-harvest sprouting-resistant red wheat.
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CRISPR/Cas9 介导的 Tamyb10 修复，创造出收获前抗发芽的红小麦。
DOI：
10.1111/pbi.13981
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发表时间：
2023-04
影响因子：
13.8
通讯作者：
Zhang, Cui-Jun
中科院分区：
文献类型：
--
作者：
Zhu, Yiwang;Lin, Yarong;Fan, Yujin;Wang, Yiwei;Li, Pengfeng;Xiong, Jiang;He, Yuhan;Cheng, Shifeng;Ye, Xingguo;Wang, Feng;Goodrich, Justin;Zhu, Jian-Kang;Wang, Ke;Zhang, Cui-Jun
小麦收获前发芽（PHS）降低产量和谷物品质，并且在世界上几乎每个小麦种植区都发生（Vetch等人，2019年）。通常，红粒小麦品种比白粒小麦品种对PHS更耐受（Himi等人，2011年）。此外，种皮的红色素含有原花青素，其抗氧化活性和自由基清除能力具有促进健康的特性。因此，培育优良红小麦品种是选育高产优质小麦的重要目标。R2 R3-MYB是植物中最大的转录因子家族之一，在调节植物发育、代谢和逆境应答等方面发挥着重要作用。六倍体小麦的R2 R3-MYB转录因子Tamyb 10激活类黄酮生物合成基因以指定红色谷粒颜色并影响PHS（Himi等人，2011年）。在大多数白色小麦品种中，Tamyb 10-A1 a、Tamyb 10-B1 a和Tamyb 10-D1 a基因具有大的插入或缺失，这破坏了IRTKAL/IRC基序和调节功能（Himi等人，2011年）。在Tamyb 10基因中，Tamyb 10-B1 a等位基因在近88.6%的面包小麦品系中具有19-bp缺失;该缺失导致开放阅读框的移码并破坏所得蛋白质（Dong et al.，2015; Himi等人，2011年）。考虑到CRISPR/Cas9诱导的突变通常是在特定靶位点处的+1/101-bp插入缺失（Zhang et al. 2014，2016），我们可以将Tamyb 10-B1 a等位基因内的移码突变（由19-bp缺失引起）恢复为框内突变（18-bp或其他三个碱基的倍数）。在这里，我们首先对春小麦栽培品种菲尔德的Tamyb 10-B1 a基因座进行测序，并确认了该白色小麦品种中Tamyb 10-B1 a第三外显子的19 bp缺失（图1a;图S1）。为了恢复Tamyb 10-B1 a等位基因的功能，设计了一种单一gRNA，以靶向19-bp缺失位点侧翼的序列（图1a;附录S1）。然后，构建pWMB 110-SpCas 9-sgRNA并通过农杆菌介导的转化转化到Fielder中（Wang et al.，2022年）。产生了15株推定的转基因植物，从中选择了5株Tamyb 10-B1 a编辑的植物（图1b）。在T0编辑的株系中，鉴定了在19-bp缺失位点上游具有1-bp插入的4株植物。这些1-bp插入通过将Tamyb 10-B1 a等位基因中的移码突变（19-bp缺失）转化为框内突变（18-bp缺失;图1b）恢复了阅读框。此外，在所有潜在脱靶位点中均未发现突变事件（表S1）。为了研究观察到的靶突变是否可遗传，我们跟踪了符合读框的Tamyb 10-B1 a变体（NF 243 -3、NF 243 -12和NF 243 -15）的遗传。T0植物中的CRISPR/Cas9诱导的突变稳定地传递到T1代，而没有发生新的突变（图1c;表S2）。序列比对分析显示，与野生型Tamyb 10-B1 b蛋白相比，符合读框的Tamyb 10-B1 a变体的蛋白序列携带6个氨基酸的微小缺失，从而恢复了完整的IRTKAL/IRC基序（图1d;图S2）。选择具有符合读码框的Tamyb 10-B1 a序列的纯合突变体来研究籽粒果皮和PHS抗性表型。如预期的，从携带框内Tamyb 10-B1 a变体的T1品系收获的谷粒的着色从白色变为红色（图1 e）。使用光谱成像室评估这些品系中花青素苷的积累。与野生型种子相比，框内Tamyb 10具有更好的抗病性。
Wheat pre-harvest sprouting (PHS) reduces yield and grain quality and occurs in almost every wheat-growing region around the world (Vetch et al., 2019). In general, red-grained wheat varieties are more tolerant to PHS than white-grained varieties (Himi et al., 2011). Moreover, the red pigment of the grain coat contains proanthocyanidins, whose antioxidant activity and free radical scavenging ability have health-promoting properties. Therefore, producing elite red wheat varieties is an important objective in breeding high-yield and high-quality wheat. R2R3-MYB is one of the largest families of transcription factors in plants, which play crucial roles in regulating plant development, metabolism, and stress responses. The R2R3-MYB transcription factor Tamyb10 of hexaploid wheat activates flavonoid biosynthesis genes to specify red grain colour and influences PHS (Himi et al., 2011). In most white wheat varieties, the Tamyb10-A1a, Tamyb10-B1a, and Tamyb10-D1a genes have large insertions or deletions, which disrupt the IRTKAL/IRC motif and regulatory function (Himi et al., 2011). Among the Tamyb10 genes, the Tamyb10-B1a allele has a 19-bp deletion in nearly 88.6% of bread wheat lines; this deletion causes a frameshift in the open-reading frame and disrupts the resulting protein (Dong et al., 2015; Himi et al., 2011). Given that CRISPR/Cas9-induced mutations are typically+ 1/À1-bp indels at the specific target sites (Zhang et al., 2014, 2016), we can revert the frameshift mutations within the Tamyb10-B1a allele (caused by a 19-bp deletion) to in-frame mutations (18-bp or other multiples of three bases). Here, we first sequenced the Tamyb10-B1a locus in the spring wheat cultivar Fielder and confirmed the 19-bp deletion in the third exon of Tamyb10-B1a in this white wheat variety (Figure 1a; Figure S1). To restore functionality of the Tamyb10-B1a allele, a single gRNA was designed to target the sequences flanking the 19-bp deletion site (Figure 1a; Appendix S1). Then, the pWMB110-SpCas9-sgRNA was constructed and transformed into Fielder via Agrobacterium-mediated transformation (Wang et al., 2022). Fifteen putative transgenic plants were produced, from which five Tamyb10-B1a-edited plants were selected (Figure 1b). Among the T0 edited lines, four plants with a 1-bp insertion upstream of the 19-bp deletion site were identified. These 1-bp insertions restored the reading frame by converting the frameshift mutation in the Tamyb10-B1a allele (19-bp deletion) to in-frame mutation (18-bp deletion; Figure 1b). Furthermore, no mutation events were found across all potential off-target sites (Table S1).To investigate whether the target mutations observed were heritable, we tracked the inheritance of in-frame Tamyb10-B1a variants (NF243-3, NF243-12, and NF243-15). The CRISPR/Cas9-induced mutations in the T0 plants were stably transmitted to the T1 generation without the occurrence of new mutations (Figure 1c; Table S2). Sequence alignment analysis showed that, compared with the wild-type Tamyb10-B1b protein, the protein sequences of the in-frame Tamyb10-B1a variants carried a minor deletion of six amino acids, thereby restoring the intact IRTKAL/IRC motif (Figure 1d; Figure S2). Homozygous mutants with the in-frame Tamyb10-B1a sequence were selected to investigate grain pericarp and PHS resistance phenotypes. As expected, the coloration of the grains harvested from T1 lines harbouring the in-frame Tamyb10-B1a variants changed from white to red (Figure 1e). The accumulation of anthocyanins in these lines was assessed using a spectral imaging chamber. Compared with the wild-type Fielder seeds, the in-frame Tamyb10 …
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DOI：
10.1111/nph.17312
发表时间：
2021-06
期刊：
The New phytologist
影响因子：
--
作者：
Lang J;Fu Y;Zhou Y;Cheng M;Deng M;Li M;Zhu T;Yang J;Guo X;Gui L;Li L;Chen Z;Yi Y;Zhang L;Hao M;Huang L;Tan C;Chen G;Jiang Q;Qi P;Pu Z;Ma J;Liu Z;Liu Y;Luo MC;Wei Y;Zheng Y;Wu Y;Liu D;Wang J
通讯作者：
Wang J
影响因子：
18
作者：
Wang, Ke;Shi, Lei;Ye, Xingguo
通讯作者：
Ye, Xingguo
影响因子：
5.4
作者：
Himi, Eiko;Maekawa, Masahiko;Noda, Kazuhiko
通讯作者：
Noda, Kazuhiko
影响因子：
13.8
作者：
Zhang, Hui;Zhang, Jinshan;Zhu, Jian-Kang
通讯作者：
Zhu, Jian-Kang
影响因子：
1.5
作者：
Dong, Zhong Dong;Chen, Jie;Cui, Dang Qun
通讯作者：
Cui, Dang Qun