Brassinosteroids (BRs) regulate various agronomic traits such as plant height, leaf angle, and grain size in rice (Oryza sativa L.); thus, BR signaling components are promising targets for molecular rational design. However, genetic materials for BR-signaling genes or family members remain limited in rice. Here, by genome editing using clustered regularly interspaced short palindromic repeats (CRSPR)/Cas9 tools, we generated a panel of single, double, triple, or quadruple mutants within three BR signaling gene families, including GSK3/SHAGGY-LIKE KINASE1 (GSK1)–GSK4, BRASSINAZOLE-RESISTANT1 (OsBZR1)–OsBZR4, and protein phosphatases with kelch-like (PPKL)1–PPKL3, under the same background (Zhonghua11, japonica). The high-order mutants were produced by either simultaneously targeting multiple sites on different genes of one family (GSKs and PPKLs) or targeting the overlapping sequences of family members (OsBZRs). The mutants exhibited a diversity of plant height, leaf angle, and grain morphology. Comparison analysis of the phenotypes together with BR sensitivity tests suggested the existence of functional redundancy, differentiation, or dominancy among the members within each family. In addition, we generated a set of transgenic plants overexpressing GSK2, OsBZR1/2, and PPKL2, respectively, in wild-type or activated forms with fusion of different tags, and also verified the protein response to BR application. Collectively, these plants greatly enriched the diversity of important agronomic traits in rice. We propose that editing of BR-related family genes could be a feasible approach for screening of desired plants to meet different requirements. Release of these materials as well as the related information also provides valuable resources for further BR research and utilization.
水稻育種逐漸進(jìn)入分子設(shè)計(jì)階段，迅速發(fā)展的基因編輯技術(shù)為不同性狀的定向改良提供了可能，然而其實(shí)際應(yīng)用仍存在局限性。即便面對(duì)同一性狀，不同育種家不同品種在不同生態(tài)區(qū)面對(duì)不同消費(fèi)人群，其改良需求也是不同的。因此，如何滿(mǎn)足多樣化動(dòng)態(tài)需求成為當(dāng)前分子設(shè)計(jì)育種的一大挑戰(zhàn)。油菜素甾醇(BR) 顯著調(diào)控水稻株型、粒型和抗性等諸多重要性狀，在作物遺傳改良及分子設(shè)計(jì)上具有巨大應(yīng)用潛力。然而，由于不同性狀之間的復(fù)雜關(guān)聯(lián)，利用BR針對(duì)某一性狀改良往往伴隨有負(fù)效應(yīng)，克服這一問(wèn)題對(duì)有效利用BR基因進(jìn)行分子設(shè)計(jì)具有重要意義。
該研究利用CRISPR/Cas9基因編輯技術(shù)，利用單載體多靶標(biāo)或針對(duì)保守序列等不同策略，分別對(duì)BR主信號(hào)通路的三個(gè)基因家族成員(GSK1-GSK4, OsBZR1-OsBZR4, PPKL1-PPKL3)進(jìn)行了混合敲除，獲得了一系列不同組合、不同位點(diǎn)的突變體，包含了豐富的性狀多樣性組合，可篩選獲得單一性狀梯度變化或各種性狀特異或多種優(yōu)異性狀組合的遺傳材料，為利用BR基因進(jìn)行分子設(shè)計(jì)以滿(mǎn)足不同改良需求提供了新思路。
此外，該研究還創(chuàng)制了GSK2、OsBZR1/2和PPKL2等代表性基因野生型或激活形式融合不同蛋白標(biāo)簽的過(guò)表達(dá)材料并進(jìn)行了分子驗(yàn)證，為后續(xù)BR相關(guān)研究及分子利用奠定了堅(jiān)實(shí)材料基礎(chǔ)。