探討LTP2基因對水稻耐鹽性狀之影響

Abstract

水稻中非專一性脂質運輸蛋白 (non-specific Lipid Transfer Protein, nsLTP) 是一類含量豐富的小分子蛋白質，具有結合與運輸各類疏水性分子功能的鹼性蛋白，能跟脂肪酸、磷脂質、乙醯輔酶A/類固醇以及芳香類化合物等分子可逆結合，根據以往的研究，OsLTP2-RNAi植株 (基因表現量降低60%以上) 雖然可以表現出比WT (Wild type) 更高的耐鹽性，但會造成株高降低以及稔實率與千粒重大減，為解決此問題，本研究利用CRISPR/Cas9對OsLTP2啟動子進行多重基因編輯，造成多位點序列缺失與插入，使不同基因編輯系有不同程度的OsLTP2表現量下降或上升，再從中篩選出可以增加耐鹽性、但株高及產量不受影響的植株。在 T0 世代中，啟動子編輯植株呈現多樣的編輯型態，OsLTP2 的基因表現量亦呈現不同程度的上升或下降，從中分別選取三個表現量下降的編輯植株 L2、L3 與 L5 (基因表現量下降百分率分別為-31.9%、-18.1%和-15.6%) 以及表現量上升的植株 U1、U2 與 U3 (基因表現量上升百分率分別為252.1%、119.3%和94.6%) 進行後續鹽分處理試驗，半量 MS 含鹽培養基下的幼苗耐鹽性測試結果顯示，L3 與 L5 具備較佳的生長表型表現，而在水耕試驗中，L2、L3、L5 與 U2 於鹽分逆境下展現出相對優異的耐鹽性，且乾重下降百分率顯著低於 WT，進一步進行生理指標分析，發現在鹽分逆境下 L3 與 L5具有較好的光合作用效率 (Fv/Fm)，葉片 DAB 染色亦顯示其過氧化氫含量較 WT 低；在正常生長條件下，L3 的株高與 WT 相近，而 L5 的株高明顯低於 WT，為探討啟動子編輯對農藝性狀之影響，選擇基因表現量上升 (U2) 與下降 (L5) 之代表性植株進行分析，結果顯示兩者在成熟期皆顯著矮化，在分蘗數方面，U2 顯著低於 WT，L5 則顯著高於 WT；產量方面，L5 與 WT 間無顯著差異，顯示其具備良好的耐鹽及產量維持能力，本研究成功透過 OsLTP2 啟動子基因編輯建立兼具耐鹽性且不影響生長或產量的基因編輯植株，提供未來耐鹽性育種研究與應用之參考。

Non-specific lipid transfer proteins (nsLTPs) in rice represent a class of abundant, small, basic proteins that function in the binding and transport of various hydrophobic molecules. According to previous studies, OsLTP2-RNAi plants, in which OsLTP2 expression is reduced by more than 60%, exhibit enhanced salt tolerance compared to wild-type (WT) plants; however, they also display significantly reduced plant height, fertility rate, and 1000-grain weight, compromising their agronomic value. To address this issue, this study utilized the CRISPR/Cas9 system to perform multiplex genome editing of the OsLTP2 promoter region, resulting in various insertions and deletions that led to differing levels of OsLTP2 expression, either upregulated or downregulated, among the edited lines. The goal was to identify gene-edited lines with improved salt tolerance but without negative impacts on plant height or yield. In the T0 generation, the promoter-edited plants displayed diverse editing patterns, resulting in a range of OsLTP2 expression levels. Six representative lines were selected for further salt stress analysis: three with reduced expression levels—L2, L3, and L5 (expression reduction rates of −31.9%, −18.1%, and −15.6%, respectively), and three with increased expression levels—U1, U2, and U3 (expression increase rates of 252.1%, 119.3%, and 94.6%, respectively). Under salt stress using half-strength MS medium, L3 and L5 exhibited superior seedling growth phenotypes. In hydroponic experiments, L2, L3, L5, and U2 demonstrated improved salt tolerance compared to WT, as indicated by significantly smaller reductions in dry weight. Further physiological assessments revealed that L3 and L5 had higher maximum photochemical efficiency (Fv/Fm) and lower hydrogen peroxide accumulation based on DAB staining under salt stress. Under normal growth conditions, L3 had comparable plant height to WT, while L5 was significantly shorter. To evaluate agronomic traits, U2 (upregulated expression) and L5 (downregulated expression) were selected. Both lines showed significantly shorter plant height at maturity. In terms of tiller number, U2 had fewer tillers than WT, whereas L5 had significantly more. Yield analysis showed no significant difference between L5 and WT, suggesting that L5 maintains good salt tolerance without compromising yield performance. In conclusion, this study successfully applied promoter editing of OsLTP2 to modulate its gene expression levels, and identified lines exhibiting enhanced salt tolerance without compromising growth or yield.