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山苍子(Litsea cubeba (Lour) Pers.)为樟科(Lauraceae)木姜子属(Litsea Lam.)落叶灌木或小乔木,是我国重要的香料植物资源之一。其植物精油具有特殊的芳香气味及抗菌、抗虫、抗氧化等多种特征,被广泛用于香料、化妆品、医药行业[1-3]。随着生物学技术的发展及山苍子基因组图谱的完成,有助于解读山苍子中基因在各个组织、器官、个体在不同发育时期表达差异,全面理解基因分子生物学功能[4-5]。开展山苍子基因功能研究,将山苍子中重要的功能基因导入到山苍子植株中进行表型鉴定和挖掘,已成为一种主要方法,其中组培再生体系和遗传转化体系的建立是重要研究内容。
植物再生体系是遗传转化的基础,不同外植体再生能力有较大差异,尤其是木本植物生长周期长、再生困难,通过调整外源激素比例可提高其再生效率[6-9]。目前,有关山苍子的腋芽器官离体再生和愈伤诱导研究已有一定的进展[10-13],但由于遗传转化体系的缺乏,利用基因工程技术进行山苍子遗传改良的研究尚未展开。农杆菌介导法是植物遗传转化采用最多的一种方法,具有成本低、操作简便、可重复性好等优点。农杆菌介导遗传转化法原理:植物受伤部位产生酚类物质(乙酰丁香酮等),在酚类物质诱导下农杆菌聚集和vir诱导因子表达,促进T-DNA复合物产生,然后T-DNA 复合物转移并整合到植物细胞的基因组中[14-15]。遗传转化步骤主要包括农杆菌液准备、侵染和共培养、转化组织的筛选和分化、转化植株的检测[16-19]。在筛选培养阶段至少需要添加两种抗生素进行筛选,一种能抑制农杆菌生长,另一种能杀死未转化细胞并保留转化成功的细胞。头孢霉素能显著抑制农杆菌生长,对植物生长影响较小,而潮霉素能显著杀死未转化的植物细胞组织,大幅降低假阳性的产生,这两种抗生素被广泛应用于植物的遗传转化[20-22]。然而,如抗生素浓度使用过度,会影响愈伤组织的正常生长和分化,甚至造成已转化成功愈伤组织的死亡,且不同植物对抗生素忍耐性不同[23]。因此,选择合适的抗生素浓度能显著提升遗传转化的效率。
本研究以快繁的无菌组培苗为试验材料,进一步优化山苍子组培再生过程,研究头孢霉素和潮霉素对山苍子再生的影响,初步建立山苍子的遗传转化体系,为山苍子种质资源繁育及基因功能研究和遗传改良提供技术支撑。
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诱导培养21 d的山苍子愈伤组织置于6-BA、IBA、TDZ 3种不同激素配比培养基下培养。试验显示:6-BA浓度在1.0~2.0 mg·L−1时的愈伤组织生长状态较好,且能保持持续分裂;IBA浓度在0.1 mg·L−1左右时有利于愈伤组织的增殖继代,而在0.01 mg·L−1 IBA低浓度时有利于愈伤组织的分化;TDZ有助于细胞生长及分裂,在加入0.05~0.50 mg·L−1 TDZ后,愈伤组织表现出持续生长和分化,有效防止了褐化。外源激素组合为1.0 mg·L−1 6-BA + 0.1 mg·L−1 IBA + 0.5 mg·L−1 TDZ有利于愈伤增殖继代培养, 愈伤组织仅在2.0 mg·L−16-BA + 0.01 mg·L−1 IBA + 0.05 mg·L−1 TDZ激素组合下出现分化,分化率在16.67%~36.67%之间(表1)。
表 1 外源激素对山苍子愈伤组织分化的影响
Table 1. Effects of exogenous hormones on callus differentiation in L. cubeba
6-BA/
(mg·L−1)IBA/
(mg·L−1)TDZ/
(mg·L−1)愈伤组织生长状态
Callus growth state6-BA/
(mg·L−1)IBA/
(mg·L−1)TDZ/
(mg·L−1)愈伤组织生长状态
Callus growth state0.1 0.01 0.01 褐化严重
Serious browning1.0 0.01 0.10 褐化严重
Serious browning0.1 0.01 0.05 褐化严重
Serious browning1.0 0.01 0.20 轻褐化
Little browning0.1 0.01 0.10 褐化严重
Serious browning1.0 0.01 0.50 轻褐化
Little browning0.1 0.01 0.20 褐化严重
Serious browning1.0 0.05 0.01 轻褐化
Little browning0.1 0.01 0.50 褐化严重
Serious browning1.0 0.05 0.05 轻褐化
Little browning0.1 0.05 0.01 褐化严重
Serious browning1.0 0.05 0.10 轻褐化
Little browning0.1 0.05 0.05 褐化严重
Serious browning1.0 0.05 0.20 轻褐化
Little browning0.1 0.05 0.10 褐化严重
Serious browning1.0 0.05 0.50 轻褐化
Little browning0.1 0.05 0.20 褐化严重
Serious browning1.0 0.10 0.01 轻褐化
Little browning0.1 0.05 0.50 褐化严重
Serious browning1.0 0.10 0.05 轻褐化
Little browning0.5 0.01 0.01 褐化严重
Serious browning1.0 0.10 0.10 淡黄绿色,致密
Yellowish green, compact0.5 0.01 0.05 褐化严重
Serious browning1.0 0.10 0.20 淡黄绿色,致密
Yellowish green, compact0.5 0.01 0.10 褐化严重
Serious browning1.0 0.10 0.50 淡黄绿色,致密
Yellowish green, compact0.5 0.01 0.20 褐化严重
Serious browning2.0 0.01 0.01 淡黄绿色,致密
Yellowish green, compact0.5 0.01 0.50 褐化严重
Serious browning2.0 0.01 0.05 淡黄绿色,致密,分化率16.67%~36.67%
Yellowish green, compact,
differentiation rate 16.67%~36.67%0.5 0.05 0.01 褐化严重
Serious browning2.0 0.01 0.10 淡黄绿色,致密
Yellowish green, compact0.5 0.05 0.05 褐化严重
Serious browning2.0 0.01 0.20 淡黄绿色,致密
Yellowish green, compact0.5 0.05 0.10 褐化严重
Serious browning2.0 0.01 0.50 淡黄绿色,致密
Yellowish green, compact0.5 0.05 0.20 褐化严重
Serious browning2.0 0.05 0.01 褐化严重
Serious browning0.5 0.05 0.50 褐化严重
Serious browning2.0 0.05 0.05 褐化严重
Serious browning0.5 0.10 0.01 褐化严重
Serious browning2.0 0.05 0.10 褐化严重
Serious browning0.5 0.10 0.05 褐化严重
Serious browning2.0 0.05 0.20 褐化严重
Serious browning0.5 0.10 0.10 褐化严重
Serious browning2.0 0.05 0.50 褐化严重
Serious browning0.5 0.10 0.20 褐化严重
Serious browning2.0 0.10 0.01 褐化严重
Serious browning0.5 0.10 0.50 褐化严重
Serious browning2.0 0.10 0.05 褐化严重
Serious browning1.0 0.01 0.01 褐化严重
Serious browning2.0 0.10 0.10 褐化严重
Serious browning1.0 0.01 0.05 褐化严重
Serious browning2.0 0.10 0.20 褐化严重
Serious browning -
将长至高3~5 cm的山苍子无菌苗去除基部愈伤组织,置于不同浓度的NAA、IBA、IAA配比下进行生根诱导。结果表明:NAA不能诱导苗生根,而IBA在适宜浓度下能诱导苗生根,但生根率较低,且基部有愈伤形成,阻碍了养分的运输及根的形成,导致叶片掉落,苗生长状态差。在培养基中仅添加IAA时植株生长状态较好,基部无愈伤组织形成,在0.5 mg·L−1 IAA时生根率达97.33%(表2)。
表 2 外源激素对山苍子组培苗生根的影响
Table 2. Effects of exogenous hormones on rooting of cultured L. cubeba seedlings
NAA/
(mg·L−1)IBA/
(mg·L−1)IAA/
(mg·L−1)生根率
Rooting rate/%植株状态
Plant growth state0.0 0.2 0.0 0.00 叶片掉落,基部长少量愈伤
Leaf all, little callus in root bases0.0 0.5 0.5 0.00 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases0.0 0.5 1.0 0.00 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases0.0 0.5 1.0 28.67±3.06 叶片掉落,基部长少量愈伤
Leaf all, little callus in root bases0.0 0.8 0.2 49.33±4.16 叶片掉落,基部长少量愈伤
Leaf all, little callus in root bases0.0 1.0 0.0 14.67±1.15 叶片掉落,基部长少量愈伤
Leaf all, little callus in root bases0.0 0.0 0.5 97.33±2.31 正常生根,基部不长愈伤
Normal rooting, no callus in root bases0.0 0.0 1.0 79.33±5.03 正常生根,基部不长愈伤
Normal rooting, no callus in root bases0.1 0.5 0.0 0.00 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases0.1 1.0 0.0 0.00 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases0.5 0.0 0.0 12.67±1.15 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases -
头孢霉素能抑制农杆菌的生长,浓度范围一般控制在200~500 mg·L−1,浓度低于200 mg·L−1不能有效抑制农杆菌的生长,浓度高于500 mg·L−1时影响愈伤的生长及分化。本试验在0、200、300、500 mg·L−1不同头孢霉素浓度下诱导茎段愈伤,在浓度200~300 mg·L−1时愈伤组织颜色鲜绿保持正常,与不添加头孢霉素保持一致。在浓度500 mg·L−1时愈伤组织颜色比正常愈伤更为深绿,出现老化状态。因此,筛选培养前期选择头孢霉素浓度300 mg·L−1作为农杆菌抑制浓度,后期应不断降低头孢霉素浓度,防止对愈伤分化产生影响(表3)。
表 3 不同浓度头孢霉素对愈伤组织诱导及生长的影响
Table 3. Effects of different concentrations of cefotaxime on callus induction and growth
头孢霉素浓度
Cefotaxime
concentrations/
(mg·L−1)愈伤诱导率
Callus induction
rate/%愈伤生长状态
Callus growth
state0 100 颜色鲜绿
Fresh green200 100 颜色鲜绿
Fresh green300 100 颜色鲜绿
Fresh green500 100 颜色深绿
Dark green -
潮霉素对愈伤组织诱导及生长影响较大,潮霉素浓度在5 mg·L−1时,能很大程度地诱导愈伤组织,但愈伤组织整体状态差且诱导培养3周后愈伤组织全部褐化;随着潮霉素浓度的增加,其浓度10 mg·L−1时已不能诱导愈伤组织(表4)。将继代培养1周后的愈伤组织在含潮霉素培养基中培养3周,随着潮霉素浓度的增加愈伤组织死亡越多,10 mg·L−1浓度下愈伤组织出现大面积褐化死亡,浓度30 mg·L−1的愈伤组织表面已无可见愈伤。因此,综合考虑遗传转化过程中多步骤操作及农杆菌对愈伤的影响,以潮霉素浓度5 mg·L−1为第1次筛选浓度,持续时间为7~10 d,潮霉素浓度20~30 mg·L−1 作为愈伤组织临界筛选浓度,持续时间应不短于2个月。
表 4 潮霉素对山苍子愈伤组织诱导及生长的影响
Table 4. Effects of hygromycin on callus induction and growth of L. cubeba
潮霉素浓度/
(mg·L−1)
Hygromycin
concentrations愈伤组织诱导
Callus induction愈伤组织生长
Callus growth0 100%成活,鲜绿色
100% survival, fresh green100%成活
100% survival5 100%成活,暗淡
100% survival, with dull callus80%成活,愈伤暗淡
80% survival, with dull callus10 无愈伤,逐渐褐化
No callus, gradually browning15%成活,大面积褐化
15% survival, many browning20 无愈伤,逐渐褐化
No callus, gradually browning5%成活,大面积褐化
5% survival, many browning30 全部褐化死亡
All browning death全部褐化死亡
All browning death50 全部褐化死亡
All browning death全部褐化死亡
All browning death100 全部褐化死亡
All browning death全部褐化死亡
All browning death -
山苍子农杆菌遗传转化流程见图1。首先在光照条件下诱导愈伤组织约21 d,并继代培养7~10 d,然后采用农杆菌侵染继代培养后的愈伤组织30 min,放置于无菌滤纸上超净台吹干至愈伤组织表面无水分;将侵染后愈伤组织转移至共培养基黑暗共培养2~3 d至刚好肉眼可见农杆菌,立即用头孢霉素清洗愈伤组织并吹干,此处应完全吹干至愈伤组织表面发白为止,防止农杆菌污染;转移愈伤组织至含5 mg·L−1潮霉素筛选培养基初步筛选抗性愈伤组织30 d左右,再将愈伤组织转移至含30 mg·L−1潮霉素进行继代培养约60 d;最后将新长出的抗性愈伤组织在10 mg·L−1潮霉素下进行抗性愈伤组织分化约30~60 d,并将诱导出的芽继代培养约60~90 d。从农杆菌侵染愈伤组织至产生抗性幼苗需要约7~9个月时间。
阳性苗的验证首先采用PCR检测目的基因的方法,共检测了16株抗性幼苗,利用第1对和第2对引物均扩增出目的条带,送北京擎科生物科技有限公司测序拼接后,与插入基因和载体的序列比对完全正确和无移码突变,表明目的基因已插入山苍子基因组中(图2)。且通过第3对引物扩增未扩增出条带,说明没有农杆菌质粒的干扰,排除了假阳性的存在(图2)。共计转化了300个左右愈伤组织,得到约80个抗性愈伤,共2个愈伤分化,得到38株幼苗,转化率为0.67%。
PCR结果主要是利用特异性引物的聚合酶链式反应,可能存在一定的假阳性。而Southern杂交法是当前鉴定外源基因整合及表达的权威方法,具有灵敏性高、特异性强、假阳性低的优点。因此,通过Southern杂交试验进一步验证目的基因是否插入山苍子基因组中。结果表明,转化的抗性愈伤组织中有阳性条带,外源基因已插入山苍子基因组中(图3)。
农杆菌介导的山苍子遗传转化体系的构建
Establishment of Agrobacterium Mediated Genetic Transformation System of Litsea cubeba
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摘要:
目的 筛选适合山苍子愈伤组织分化的激素比例,研究其对抗生素的耐受性,构建山苍子遗传转化体系。 方法 以山苍子无菌苗茎段诱导的愈伤组织为试验材料,研究不同激素浓度配比对山苍子愈伤组织诱导不定芽及不定芽生根的影响,探讨愈伤组织对潮霉素和头孢霉素的临界筛选浓度,并通过农杆菌介导法将外源基因导入山苍子愈伤组织中。 结果 筛选出诱导愈伤组织不定芽分化培养基为:MS + 2.0 mg·L−1 6-BA + 0.01 mg·L−1 IBA + 0.05 mg·L−1 TDZ,分化率为16.67%~36.67%;不定芽生根培养基为1/2MS + 0.5 mg·L−1 IAA,生根率为97.33%。遗传转化初期筛选抗性愈伤组织的潮霉素浓度为5 mg·L−1(约7~10 d),通过逐步增加潮霉素浓度至30 mg·L−1进行筛选培养,头孢霉素最适浓度为300 mg·L−1。采用农杆菌介导法将外源基因转入愈伤组织中,并设计PCR引物进行鉴定,共检测16株抗性苗均含有目的条带,表明目的基因已插入山苍子基因组中,转化率为0.67%。通过Southern检测表明,目的片段已插入山苍子愈伤组织中。 结论 初步建立山苍子再生及遗传转化体系,且已获得多个基因的抗性愈伤组织,为进一步开展基因功能研究及遗传改良提供技术支持。 Abstract:Objective To screen the hormone proportion suitable for callus differentiation of Litsea cubeba and clarify its tolerance to antibiotics, and preliminarily establish the genetic transformation system of L. cubeba. Method The effects of different hormone concentrations on adventitious bud induction and adventitious bud rooting of L. cubeba callus were studied. The critical screening concentrations of hygromycin and cefotaxime were discussed, and the foreign gene was introduced into L.cubeba callus by Agrobacterium mediated method. Result The optimum medium for inducing adventitious bud differentiation of callus was MS + 2.0 mg·L−1 6-BA + 0.01 mg·L−1 IBA + 0.05 mg·L−1 TDZ, and the differentiation rate was 16.67%~36.67%; The optimum medium for adventitious bud rooting was 1/2MS + 0.5 mg·L−1 IAA, and the rooting rate was 97.33%. The initial concentration of hygromycin for resistant callus screening was 5 mg·L−1 (about 7~10 days), and then the critical screening culture was carried out by gradually increasing the hygromycin screening concentration to 30 mg·L−1. the optimum concentration of cephalosporin was 300 mg·L−1. Finally, foreign gene was transferred into the callus by Agrobacterium mediated method, and PCR primers were designed for identification. A total of 16 resistant seedlings contained the target band, indicating that the target gene had been inserted into the L. cubeba genome, with a transformation rate of 0.67%. In addition, our research group obtained the resistant calli of multiple genes through this method. Southern detection showed that the target fragment had been inserted into the calli of L. cubeba. Conclusion The regeneration and genetic transformation system of L. cubeba has been preliminarily established, and the resistant calli of multiple genes have been obtained, which provides technical support for further gene function research and genetic improvement. The next step is to optimize the genetic transformation system and improve its transformation efficiency. -
Key words:
- Litsea cubeba
- / regeneration
- / antibiotic
- / genetic transformation
- / Agrobacterium mediated method
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表 1 外源激素对山苍子愈伤组织分化的影响
Table 1. Effects of exogenous hormones on callus differentiation in L. cubeba
6-BA/
(mg·L−1)IBA/
(mg·L−1)TDZ/
(mg·L−1)愈伤组织生长状态
Callus growth state6-BA/
(mg·L−1)IBA/
(mg·L−1)TDZ/
(mg·L−1)愈伤组织生长状态
Callus growth state0.1 0.01 0.01 褐化严重
Serious browning1.0 0.01 0.10 褐化严重
Serious browning0.1 0.01 0.05 褐化严重
Serious browning1.0 0.01 0.20 轻褐化
Little browning0.1 0.01 0.10 褐化严重
Serious browning1.0 0.01 0.50 轻褐化
Little browning0.1 0.01 0.20 褐化严重
Serious browning1.0 0.05 0.01 轻褐化
Little browning0.1 0.01 0.50 褐化严重
Serious browning1.0 0.05 0.05 轻褐化
Little browning0.1 0.05 0.01 褐化严重
Serious browning1.0 0.05 0.10 轻褐化
Little browning0.1 0.05 0.05 褐化严重
Serious browning1.0 0.05 0.20 轻褐化
Little browning0.1 0.05 0.10 褐化严重
Serious browning1.0 0.05 0.50 轻褐化
Little browning0.1 0.05 0.20 褐化严重
Serious browning1.0 0.10 0.01 轻褐化
Little browning0.1 0.05 0.50 褐化严重
Serious browning1.0 0.10 0.05 轻褐化
Little browning0.5 0.01 0.01 褐化严重
Serious browning1.0 0.10 0.10 淡黄绿色,致密
Yellowish green, compact0.5 0.01 0.05 褐化严重
Serious browning1.0 0.10 0.20 淡黄绿色,致密
Yellowish green, compact0.5 0.01 0.10 褐化严重
Serious browning1.0 0.10 0.50 淡黄绿色,致密
Yellowish green, compact0.5 0.01 0.20 褐化严重
Serious browning2.0 0.01 0.01 淡黄绿色,致密
Yellowish green, compact0.5 0.01 0.50 褐化严重
Serious browning2.0 0.01 0.05 淡黄绿色,致密,分化率16.67%~36.67%
Yellowish green, compact,
differentiation rate 16.67%~36.67%0.5 0.05 0.01 褐化严重
Serious browning2.0 0.01 0.10 淡黄绿色,致密
Yellowish green, compact0.5 0.05 0.05 褐化严重
Serious browning2.0 0.01 0.20 淡黄绿色,致密
Yellowish green, compact0.5 0.05 0.10 褐化严重
Serious browning2.0 0.01 0.50 淡黄绿色,致密
Yellowish green, compact0.5 0.05 0.20 褐化严重
Serious browning2.0 0.05 0.01 褐化严重
Serious browning0.5 0.05 0.50 褐化严重
Serious browning2.0 0.05 0.05 褐化严重
Serious browning0.5 0.10 0.01 褐化严重
Serious browning2.0 0.05 0.10 褐化严重
Serious browning0.5 0.10 0.05 褐化严重
Serious browning2.0 0.05 0.20 褐化严重
Serious browning0.5 0.10 0.10 褐化严重
Serious browning2.0 0.05 0.50 褐化严重
Serious browning0.5 0.10 0.20 褐化严重
Serious browning2.0 0.10 0.01 褐化严重
Serious browning0.5 0.10 0.50 褐化严重
Serious browning2.0 0.10 0.05 褐化严重
Serious browning1.0 0.01 0.01 褐化严重
Serious browning2.0 0.10 0.10 褐化严重
Serious browning1.0 0.01 0.05 褐化严重
Serious browning2.0 0.10 0.20 褐化严重
Serious browning表 2 外源激素对山苍子组培苗生根的影响
Table 2. Effects of exogenous hormones on rooting of cultured L. cubeba seedlings
NAA/
(mg·L−1)IBA/
(mg·L−1)IAA/
(mg·L−1)生根率
Rooting rate/%植株状态
Plant growth state0.0 0.2 0.0 0.00 叶片掉落,基部长少量愈伤
Leaf all, little callus in root bases0.0 0.5 0.5 0.00 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases0.0 0.5 1.0 0.00 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases0.0 0.5 1.0 28.67±3.06 叶片掉落,基部长少量愈伤
Leaf all, little callus in root bases0.0 0.8 0.2 49.33±4.16 叶片掉落,基部长少量愈伤
Leaf all, little callus in root bases0.0 1.0 0.0 14.67±1.15 叶片掉落,基部长少量愈伤
Leaf all, little callus in root bases0.0 0.0 0.5 97.33±2.31 正常生根,基部不长愈伤
Normal rooting, no callus in root bases0.0 0.0 1.0 79.33±5.03 正常生根,基部不长愈伤
Normal rooting, no callus in root bases0.1 0.5 0.0 0.00 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases0.1 1.0 0.0 0.00 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases0.5 0.0 0.0 12.67±1.15 叶片掉落,基部长大量愈伤
Leaf all, many callus in root bases表 3 不同浓度头孢霉素对愈伤组织诱导及生长的影响
Table 3. Effects of different concentrations of cefotaxime on callus induction and growth
头孢霉素浓度
Cefotaxime
concentrations/
(mg·L−1)愈伤诱导率
Callus induction
rate/%愈伤生长状态
Callus growth
state0 100 颜色鲜绿
Fresh green200 100 颜色鲜绿
Fresh green300 100 颜色鲜绿
Fresh green500 100 颜色深绿
Dark green表 4 潮霉素对山苍子愈伤组织诱导及生长的影响
Table 4. Effects of hygromycin on callus induction and growth of L. cubeba
潮霉素浓度/
(mg·L−1)
Hygromycin
concentrations愈伤组织诱导
Callus induction愈伤组织生长
Callus growth0 100%成活,鲜绿色
100% survival, fresh green100%成活
100% survival5 100%成活,暗淡
100% survival, with dull callus80%成活,愈伤暗淡
80% survival, with dull callus10 无愈伤,逐渐褐化
No callus, gradually browning15%成活,大面积褐化
15% survival, many browning20 无愈伤,逐渐褐化
No callus, gradually browning5%成活,大面积褐化
5% survival, many browning30 全部褐化死亡
All browning death全部褐化死亡
All browning death50 全部褐化死亡
All browning death全部褐化死亡
All browning death100 全部褐化死亡
All browning death全部褐化死亡
All browning death -
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