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杨树是我国短周期工业用材林的主栽树种之一。近年来,随着人工林面积的增加及品种单一化,杨树的抗病性显著下降。杨树溃疡病是危害杨树主要枝干病害之一[1],是由葡萄座腔菌属(Botryosphaeria)、腐皮壳菌属(Valsa)和壳梭孢属(Fusicoccum)等病原真菌侵染引起的。在我国,杨树溃疡病主要是由葡萄座腔菌属引起的水泡型溃疡病[2]。水泡型溃疡病在东北、华北、西北等地区大面积流行,给杨树生产造成了极大的损失。化学手段防治杨树溃疡病不仅收效甚微且费用高,还可能污染环境。因此,培育抗病品种已成为当前防治杨树溃疡病的重要途径[3]。
研究表明,将抗病基因转入杨树,能够显著提高杨树的抗病性。例如,转无色花色素还原酶基因(PtrLAR3)的毛白杨对杨盘二孢菌的抗性显著增强[4];过表达WRKY转录因子(PtoWRKY60)基因及过表达拟南芥MYB115基因的转基因毛白杨对溃疡病的抗性明显增强[5-6];转毛白杨WRKY转录因子基因(PtrWRKY18和PtrWRKY35)的毛白杨对病原菌栅锈菌的抗性明显提高[7]。
库安托杨(Populus × euramericana ‘Guariento’)是我国从意大利引进的优良品种,但其抗溃疡病等枝干病害的能力较差。2006年,王建革等[8]将5个外源基因(JERF36、SacB、vgb、BtCry3A、OC-1)转入库安托杨,获得了对多种逆境胁迫均具有较强抗性的转基因株系[9-12]。在5个转入的外源基因中,JERF36是从野生番茄中克隆的ERF (ethylene responsive factor,乙烯应答因子)类转录因子基因[13],在抗病信号传导过程中起重要的作用[14],但其在杨树抗病性中的作用尚未见报道。
本研究对1年生转多基因库安托杨株系(D5-19、D5-20、D5-21)及非转基因对照株系(D5-0)进行了杨树溃疡病菌接种试验,检测了树皮中5个抗病性相关基因的表达,对转多基因库安托杨溃疡病抗性进行了综合评价,为其推广应用奠定了基础,也为其它林木抗病分子育种提供参考。
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植物材料为3个转多基因库安托杨株系(D5-19、D5-20和D5-21)及未转基因的对照株系(D5-0)。于2016年3月,选择越冬后植株高度在2 m左右、地径2.5 cm左右的1年生植株,从植株基部往上截取1.5 m的主干,将新截取的主干基部插入自来水中进行水培,培养条件为全自动控温日光温室,每天更换自来水,60 d后进行溃疡病病原菌接种。
接种菌株为葡萄座腔菌(Botryosphaeria dothidea)CZA菌株(保存于中国林科院林业新技术研究所)。菌株活化培养后,接种于PDA培养基进行暗培养,在28℃条件下培养10 d,取直径0.9 cm菌饼用于杨树主干接种。
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距截取处30 cm高处开始,每隔约10 cm在枝干上均匀打孔,深度至露出木质部,打孔直径为0.9 cm,每个枝条打孔1012个。在打孔处接种杨树溃疡病CZA菌株菌饼,接种后用Parafilm密封接种处,同时在主干顶部封蜡以防止枝条失水。以打孔但未接种菌株的枝条(CK)作为对照,接种菌株枝条作为处理,对照和处理均设置5株作为生物学重复,试验重复3次。在接种后5 d后取接种孔间的健康树皮,液氮冷冻后置于超低温冰箱保存,用于提取总RNA。接种处理后第60天观察打孔处愈伤组织形成情况,并测量纵向溃疡斑大小。
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参考黄逢龙等对杨树溃疡病病情指数的计算方法[2],结合本研究中苗木的发病状况,依据形成病斑的纵向长度,将病斑划分为5个不同的级别(表 1)。统计不同株系数据,计算杨树溃疡病病情指数。计算公式为:病情指数=[∑(各级病斑数×各级代表值)/(总接种点数×5)]×100。数据分析采用SPSS 19.0软件。
表 1 杨树溃疡病病级划分标准
Table 1. Classification standard of poplar canker disease
病级
Cankerrank代表值
Representative value分类标准
Standard of classificationⅠ 0 形成愈伤组织或不形成病斑 Ⅱ 1 病斑≤1.0 cm Ⅲ 2 1.0 cm < 病斑≤2.0 cm Ⅳ 3 2.0 cm < 病斑≤3.0 cm Ⅴ 4 3.0 cm < 病斑≤4.0 cm Ⅵ 5 4.0 cm < 病斑 -
采用Qiangen RNeasy Plant Mini Kit提取接种点间健康树皮组织的总RNA。以Promega Super-Script TM Ⅲ第一链合成试剂盒反转录合成cDNA,并稀释10倍后用于RT-qPCR分析。使用Takara SYBR Premix Ex Taq试剂盒配制RT-qPCR反应体系,其中,cDNA模板体积为1.0 μL,其他成分为试剂盒推荐浓度。在Roche LightCycle 480Ⅱ型荧光定量PCR仪上对目标基因进行扩增,每个样品设置4个反应重复。选取杨树UBQ-like作为内参基因,抗病基因信息见表 2。PCR扩增程序为:预变性95℃,5 min;变性95℃,10 s,退火60℃,10 s,延伸72℃,10 s,45个循环。抗性基因的相对表达水平采用2-△△CT法进行计算[15]。
表 2 抗病基因及内参基因引物实时定量PCR引物
Table 2. List of amplified genes and primer sequence
基因名称Gene name 引物序列Primer sequence 产物大小Product size/bp 几丁质酶基因
Chitinase gene5’-CCTCATGCCACAATGTCATC-3’
5’-CGATCCTCCACCTGTGATTT-3’148 链蛋白A基因
Chain A gene5’-TGGGTCTTCACGGTTTTCTC-3’
5’-ACATAGACCTCCCGGACACA-3’105 肉桂醇脱氢酶基因
Cinnamyl-alcohol dehydrogenase gene5’-CGTCAAGGTGACGGTGATTA-3’
5’-TCACGACTGACCAAAAACGA-3’90 病程相关蛋白基因
Pathogenesis-related protein gene5’-GCGATTGCAACCTTATCCAT-3’
5’-GCCTTCTCATCAACCCACAT-3’109 类甜蛋白基因
Thaumatin-like gene5’-GTATTGGTGGTGGTGCTCCT-3’
5’-AGCCAACATTGTAGCCATCC-3’111 内参基因
UBQ-Like5’-TGAGGCTTAGGGGAGGAACT-3’
5’-TGTAGTCGCGAGCTGTCTTG-3’195 -
接种溃疡病菌株60 d后,除去接种点的Parafilm,测量各株系枝条上的病斑纵向长度(图 1),将病斑数据换算为各病级和各病级代表值后,根据1.3中的病情指数公式计算各株系的病情指数,采用SPSS 19.0软件进行显著性差异检测。
图 1 溃疡病菌接种60 d后对照及转基因株系的病斑
Figure 1. The canker spots in bark of control and transgenic clones after B. dothidea inoculation for 60 days
实验结果(图 1)显示:非转基因对照株系D5-0的病情指数为27.59±1.36,转基因株系D5-19、D5-20和D5-21的病情指数分别为22.75±1.37、22.19±1.53和20.12±0.21。Fisher’s LSD检验结果(表 3)表明:对照株系病情指数极显著高于3个转基因株系,3个转基因株系中,D5-19与D5-20的病情指数间差异不显著,而D5-21的病情指数显著低于D5-19。抗病性数据分析结果表明:3个转基因库安托杨株系的溃疡病抗性较对照株系显著增强,同时,转基因株系间抗病性并不相同,D5-21株系对溃疡病具有更强的抗性。
表 3 转多基因杨树株系及对照溃疡病病情指数的显著性检测结果
Table 3. Significant analysis of the canker disease index of transgenic poplar clones and the control
株系
Clone病情指数
Disease index平均病情指数
Average disease
index显著差异性
Significant differenceT01 T02 T03 5% 1% D5-0 28.00 28.70 26.07 27.59±1.36 a A D5-19 22.41 24.26 21.59 22.75±1.37 b B D5-20 20.62 23.69 22.26 22.19±1.53 bc B D5-21 20.37 20.00 20.00 20.12±0.21 c B -
本研究对溃疡病病原菌接种5 d后,对照及转基因株系树皮中5个抗病相关基因的表达特征进行了相对定量分析。RT-qPCR结果显示:在溃疡病菌的胁迫后,不同抗病相关基因对溃疡病菌的响应差异较大(图 2)。
图 2 溃疡病菌接种5 d后对照及转基因株系树皮中5个抗病相关基因的表达情况
Figure 2. Expression pattern of 5 disease resistant genes in bark of control and transgenic clones after B. dothidea inoculation for 5 days
接种溃疡病菌5 d后,转基因株系及对照中几丁质酶基因(图 2A)、链蛋白A基因(图 2B)及病程相关蛋白基因(图 2D)的表达情况相似,即D5-19树皮中这3个抗病基因的表达量与对照相当,而在其他2个转基因株系树皮中这3个基因的表达量均低于对照。肉桂醇脱氢酶基因(图 2C)在3个转基因株系树皮中表达量均高于对照,其中,D5-20株系中的表达量为对照中的1.5倍左右,D5-19中的表达量为对照的3倍左右,D5-21中表达量为对照的6倍左右。类甜蛋白基因(图 2E)在D5-19树皮中的表达量高于对照,但在其他2个转基因株系中表达量均低于对照。综合分析接种溃疡病菌5 d后对照及转基因各株系树皮中5个抗病相关基因相对表达量发现,溃疡病侵染后,只有肉桂醇脱氢酶基因在3个转基因株系树皮中表达量均比对照有较大幅度的上升,并在D5-21中表达量最高;而类甜蛋白基因只在转基因株系D5-19中表达量高于对照。因此,转基因株系抗病性的提高与类甜蛋白基因及肉桂醇脱氢酶基因在转基因株系中的高表达密切相关。
转多基因1年生库安托杨对溃疡病的抗性分析
Resistance Evaluation of the One-year-old Multiple Transgenic Populus×euramericana 'Guariento' to Poplar Canker
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摘要:
目的 本研究对转多基因库安托杨株系的溃疡病抗病性检测及抗病相关基因的表达分析,为通过基因工程手段培育抗病林木新树种提供有价值的参考。 方法 以转JERF36等多个外源基因的1年生库安托杨株系D5-9、D5-20、D5-21及非转基因受体D5-0为材料,对主干人工接种溃疡病菌的各株系病情指数进行了测定,同时对接种5 d后树皮组织中5个抗病基因的表达情况进行了实时定量PCR(qPCR)分析。 结果 在溃疡病菌胁迫下,3个转基因株系对溃疡病菌的抗性显著优于非转基因受体株系,转基因株系间抗病性也具有一定差异,D5-21的病情指数显著低于其它2个转基因株系;肉桂醇脱氢酶基因在3个转基因株系树皮中表达量均高于对照,类甜蛋白基因仅在D5-19树皮中的表达量高于对照,其他3个基因在转基因株系中的表达量或与对照相当或低于对照。 结论 转多基因库安托杨株系的溃疡病抗性与非转基因对照相比均有显著提高,且转基因株系间差异显著;同时转基因株系中不同抗病基因的表达模式也有很大差异,说明溃疡病菌胁迫下转多基因库安托杨抗病调控的复杂性,其分子调控机制还有待深入研究。 Abstract:Objective To evaluate the resistance to poplar canker and mRNA expression of disease resistance-related genes of three transgenic Populus×euramericana 'Guariento' lines so as to provide reference for breeding disease-resistant trees through transgenic technology. Method Using 1-year-old stem of multiple transgenic Populus×euramericana 'Guariento' clones (D5-9, D5-20, and D5-21) and the wild control (D5-0) as material, the disease index of canker was analyzed after inoculated with Botryosphaeria dothidea for 60 days, and the expression of five disease resistance related genes in bark of these clones after inoculated with B. dothidea for 5 days were detected by relative quantitative PCR (qPCR). Result The results showed that the resistance of the three transgenic clones to poplar canker was significantly higher than that of the non-transgenic clone and obvious difference in canker resistance was observed among the three transgenic poplar clones with D5-21 was higher than the other two clones. The expression of cinnamyl-alcohol dehydrogenase family protein gene in the bark of the 3 transgenic clones was higher than that of the control, and the expression of thaumatin-like protein gene was higher than control only in bark of D5-19, whereas the expressions of the other three genes were similar to or lower than the control. Conclusion The difference in the canker resistance of the three transgenic clones and various expression profiles of the five disease resistant genes indicates the regulation complexity in the disease resistance of the multiple transgenic poplars under stress of B. dothidea, suggesting further study is needed. -
Key words:
- transgenic poplar
- / ERF
- / poplar canker
- / disease resistance
- / gene expression
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表 1 杨树溃疡病病级划分标准
Table 1. Classification standard of poplar canker disease
病级
Cankerrank代表值
Representative value分类标准
Standard of classificationⅠ 0 形成愈伤组织或不形成病斑 Ⅱ 1 病斑≤1.0 cm Ⅲ 2 1.0 cm < 病斑≤2.0 cm Ⅳ 3 2.0 cm < 病斑≤3.0 cm Ⅴ 4 3.0 cm < 病斑≤4.0 cm Ⅵ 5 4.0 cm < 病斑 表 2 抗病基因及内参基因引物实时定量PCR引物
Table 2. List of amplified genes and primer sequence
基因名称Gene name 引物序列Primer sequence 产物大小Product size/bp 几丁质酶基因
Chitinase gene5’-CCTCATGCCACAATGTCATC-3’
5’-CGATCCTCCACCTGTGATTT-3’148 链蛋白A基因
Chain A gene5’-TGGGTCTTCACGGTTTTCTC-3’
5’-ACATAGACCTCCCGGACACA-3’105 肉桂醇脱氢酶基因
Cinnamyl-alcohol dehydrogenase gene5’-CGTCAAGGTGACGGTGATTA-3’
5’-TCACGACTGACCAAAAACGA-3’90 病程相关蛋白基因
Pathogenesis-related protein gene5’-GCGATTGCAACCTTATCCAT-3’
5’-GCCTTCTCATCAACCCACAT-3’109 类甜蛋白基因
Thaumatin-like gene5’-GTATTGGTGGTGGTGCTCCT-3’
5’-AGCCAACATTGTAGCCATCC-3’111 内参基因
UBQ-Like5’-TGAGGCTTAGGGGAGGAACT-3’
5’-TGTAGTCGCGAGCTGTCTTG-3’195 表 3 转多基因杨树株系及对照溃疡病病情指数的显著性检测结果
Table 3. Significant analysis of the canker disease index of transgenic poplar clones and the control
株系
Clone病情指数
Disease index平均病情指数
Average disease
index显著差异性
Significant differenceT01 T02 T03 5% 1% D5-0 28.00 28.70 26.07 27.59±1.36 a A D5-19 22.41 24.26 21.59 22.75±1.37 b B D5-20 20.62 23.69 22.26 22.19±1.53 bc B D5-21 20.37 20.00 20.00 20.12±0.21 c B -
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