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黄芩干种子来源于定西市农业科学研究院。2022年4月,将黄芩种子置于铺有去离子水浸湿滤纸的方形培养皿中(10 cm×10 cm),置于恒温光照培养箱(24±1) ºC萌发,光周期:光照16 h加黑暗8 h。萌发9 d后,挑选长势一致的幼苗置于含有湿润滤纸的35 mm圆形培养皿中,室温下进行高能重离子束辐射处理。
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高能12C6+束流由兰州重离子研究装置(Heavy Ion Research Facility in Lanzhou, HIRFL)的浅层重离子束生物辐射终端(Terminal 4, TR4)提供,初始能量为967 MeV,贯穿样品的平均LET为34 keV·μm−1,剂量率为20 Gy·min−1,辐照剂量分别为5, 10, 20, 30和40 Gy。对照组置于和处理组相同的环境条件中,但不进行辐照处理。
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辐照后立即将幼苗移栽至50孔穴盘,培养基质为营养土:珍珠岩 = 3:1,在光照培养间中培养,光周期为光照14 h加黑暗10 h,温度为(21±2) ºC。3周后将幼苗转移至方形育苗盆中(9 cm×9 cm),继续培养至9周。
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对辐照后生长了3周的黄芩幼苗进行存活率统计,长出2对及以上真叶的幼苗记为存活植株。
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苗高:分别对辐射后生长了3周、6周和9周的黄芩幼苗进行苗高测定,以幼苗子叶基部至幼苗顶端的距离为苗高,每处理组测量10株。
叶片数:分别对辐射后生长了3周、6周和9周的黄芩幼苗进行叶片数测定,每处理组测量10株。
分枝数:对辐射后生长了9周的黄芩幼苗进行分枝数测定,每处理组测量10株。
生物量:分别对辐射后生长了3周、6周和9周的黄芩幼苗的地上和地下部分生物量分别进行测定,并计算根冠比,每处理组测量5株。
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分别对辐射后生长3周、6周和9周的黄芩幼苗近顶端第二对真叶进行混合取样0.05 g,用乙醇浸提法[26]提取叶绿素。使用酶标仪(Tecan Infinite 200)测定665、649及470 nm波长下的吸光度,根据公式计算叶片单位鲜重的Chl a、Chl b、总叶绿素(Total Chlorophyll, Total Chl)和类胡萝卜素(Carotenoids, Car)含量。计算如下:
$$ {C}_{\rm{Chl}\;{\rm{a}}} = 13.95 \times {{A}}_{\rm{665}} - 6.88 \times {{A}}_{649} \text{,} $$ $$ C_{{\rm{Chl}}{\text{ }}{\rm{b}}} = 24.96 \times {{A}}_{649} - 7.32 \times {{A}}_{665} \text{,} $$ $$ C_{{\rm{Total}}{\text{ }}{\rm{Chl}}} = 6.63 \times {A}_{665} - 18.07 \times A_{649} \text{,} $$ $$ {C}_{\rm{Car}} = \frac{{1\,\,000 \times {{A}}_{470} - 2.05 \times {{C}}_{{\rm{Chl}}{\text{ }}{\rm{a}}} - 114.8 \times {{C}}_{{\rm{Chl}}{\text{ }}{\rm{b}}}}}{{245}} \text{,} $$ $$\omega = \frac{{{{C}} \times {{V}}}}{{{W}}} \times 1\,\,000 \text{,} $$ 式中:C为光合色素浓度(mg/L);A为相应波长下的吸光度;ω为光和色素含量(mg/g);V为提取液体积(L);W为样品鲜重(g)。
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分别取辐射后生长了3周、6周和9周的黄芩幼苗地上部分叶片,混合取样0.1 g,液氮速冻后置于−80 ºC低温冰箱冷冻保存,用于后续抗氧化酶分析。SOD活性采用氮蓝四唑法[27]测定,过氧化物酶(Peroxidase, POD)活性采用愈创木酚法[28]测定,过氧化氢酶(Catalase, CAT)活性采用过氧化氢分解法[29]测定,MDA含量采用硫代巴比妥酸显色法[29]测定。
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辐射后的黄芩幼苗培养9周,使用便携式光合作用仪(Yaxin-1102G)测定幼苗叶片的净光合速率 (Net Photosynthetic Rate, Pn),气孔导度(Stomatal Conductance, Gs),蒸腾速率 (Transpiration Rate, Tr)及胞间二氧化碳浓度(Intercellular CO2 Concentration, Ci)。
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总黄酮提取:辐射后的黄芩幼苗培养9周,取材幼苗地下部分,置于烘箱中60 ºC干燥48 h,用研钵研成粉末。各组取0.1 g样品置于50 mL锥形瓶中,加入25 mL 70%乙醇,超声提取40 min,过滤,滤渣再加入25 mL 70%乙醇,超声40 min,过滤。合并两次滤液,在60 ºC下浓缩干燥。所得干燥粉末用70%乙醇定容至10 mL,待测。
标准曲线制作:配制浓度为 0.416 mg/mL的芦丁标准品95%乙醇溶液。量取 0,0.2,0.4,0.6,0.8和1.0 mL芦丁标准液,分别加入5%的亚硝酸钠水溶液0.3 mL,室温静置6 min;加入10%的硝酸铝水溶液0.3 mL,静置6 min;加入4%氢氧化钠水溶液4 mL,用去离子水定容至10 mL,充分摇匀后静置 3 min,使用酶标仪测定508 nm波长下的吸光度。得到线性回归方程为
$$ {{A}}_{508} = 5.633\,\,9C + 0.042\,\,6({R^2} = 0.998\,\,3) \text{,} $$ $$ \omega = \frac{{C \times V}}{W} \text{,} $$ 式中:A表示吸光度;C表示总黄酮浓度(mg/mL);ω表示总黄酮含量(mg/g);V表示待测液体积(L);W表示样品重量(g)。
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使用Agilent 5 TC C18色谱柱(4.6 mm×250 mm, 5 μm);流动相:0.1%甲酸水溶液(A)-乙腈(B),梯度洗脱(0~5 min, 32%~35% B; 5~15 min, 35%~50% B),流速1 mL/min;检测波长275 nm;柱温27ºC;进样量10 μL。
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称取3.94 mg黄芩素对照品(中国食品药品检定研究院,纯度97.9%,批号111595-201808)置于10 mL容量瓶中,加甲醇超声溶解并定容至10 mL。
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取1.4.6节的待测液1.5 mL,用0.22 μm微孔滤膜过滤,滤液即为供试品溶液。
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分别精密量取对照品溶液0.4, 0.8, 1.2, 1.6, 2.0 mL置于5个2 mL容量瓶中,加甲醇稀释至2 mL摇匀,0.22 μm微孔滤膜过滤,备用;分别吸取上述不同浓度的溶液各10 μL,按1.4.7.1节色谱条件进样测定。以峰面积积分值(Y)对其质量浓度(X, mg/mL)进行线性回归,得到回归方程为
$$ Y = 5.486 \times {10^7}X + 7.194 \times {10^4}({R^2} = 0.9995) \text{,} $$ 表明黄芩素在0.039~0.386 mg/mL的检测浓度范围内线性关系良好。
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所有试验均重复3次,所得数据用平均值±标准差表示。差异显著性分析使用SPSS 29软件的单因素方差分析(One-way ANOVA)的Tukey法进行。使用GraphPad Prism 9软件进行单击多靶模型拟合及绘图。
Biological Effects of High-Energy Carbon Ion Beam Irradiation on Seedlings of Scutellaria baicalensis
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摘要: 为探究高能重离子束辐射对黄芩幼苗生理特性及生长发育的影响,采用总能量为967 MeV的碳离子束辐射黄芩幼苗,测定了不同剂量辐射后的存活率、苗高、叶片数、分枝数、生物量、根冠比、抗氧化酶活性、光合作用特性及次生代谢产物的含量。结果显示,与对照组比较,黄芩幼苗的半致死剂量为32.82 Gy。5和10 Gy的重离子束辐射后黄芩的苗高、叶片数、分枝数和生物量均有增加,而30 Gy辐射均为降低。各处理组超氧化物歧化酶(Superoxide dismutase, SOD)和过氧化物酶(Peroxidase, POD)活性增加以减轻辐射造成的活性氧损伤。辐照后6周内,叶片中叶绿素含量随剂量呈下降趋势。辐照后第9周,5和10 Gy辐照提高了叶片的总叶绿素含量、净光合速率和气孔导度,10 Gy辐射促进了黄芩根中总黄酮和黄芩素的积累。研究发现,10 Gy的重离子束辐射促进了黄芩幼苗的生长,并且提高了其药用成分含量。本工作探究了重离子束辐射黄芩的当代生物学效应,为后续开展黄芩的辐射刺激效应及辐射育种研究提供了基础依据。Abstract: To explore the effects of high-energy heavy ion beams irradiation on the physiological and growth characteristics of Scutellaria baicalensis Georgi seedlings, different doses of carbon ion beam with total energy of 967 MeV were selected to irradiate seedlings to measure the survival rate, seedling height, number of leaves, number of branches, biomass, root-shoot ratio, antioxidant enzyme activity, photosynthetic characteristics and content of secondary metabolites. The results showed that the semi-lethal dose of seedlings was 32.82 Gy. The seedling height, number of leaves, number of branches, and biomass of Scutellaria baicalensis increased under 5 and 10 Gy, but decreased under 30 Gy compared with the control group. The activities of superoxide dismutase (SOD) and peroxidase (POD) in all treatment groups increased to reduce the damage of reactive oxygen species caused by irradiation. The content of chlorophyll in leaves decreased with the dose within 6 weeks after irradiation; the content of total chlorophyll, net photosynthetic rate and stomatal conductance of leaves increased under 5 and 10 Gy in 9 weeks after irradiation. In addition, the accumulation of total flavonoids and baicalein in the root were promoted under 10 Gy. These results indicated that 10 Gy heavy ion beams not only stimulated the growth, but also increased the content of medicinal ingredients in Scutellaria baicalensis. The study explored the contemporary biological effects of heavy ion beams radiation on Scutellaria baicalensis Georgi, which provides a basis for the follow-up research on irradiation stimulation effects and radiation breeding of Scutellaria baicalensis Georgi.
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Key words:
- Scutellaria baicalensis /
- carbon ion beam /
- irradiation /
- biological effect /
- antioxidant enzymes
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