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国槐作为我国的特色树种,树冠硕大,姿态优美,具有极高的园林观赏价值,在城市绿化中发挥着重要作用。国槐带化病是近年国槐苗木繁育及园林景观营造中危害比较严重的一种侵染性病害,其发病后导致国槐生长减缓,枝条扁茎、顶端小叶丛生,并最终发生枯死,不仅给园林生产带来较大的经济损失,而且近年来该病发生日趋严重,有些苗圃中国槐受害率已达30%,严重威胁园林绿化生态效益与观赏价值[1-2]。自上世纪90年代首次证实国槐带化病为植原体侵染造成以来,国内外对其关注较少且研究并不深入。直到近年随着分子检测技术的快速发展,杜银银[3]通过采用PCR快速扩增及生物信息学技术首次明确了国槐带化病植原体的分类地位,并对国槐带化病植原体2个株系(16Sr I-D与16Sr I-B)之间的遗传相关性和亲缘关系进行了鉴定分析。但由于植原体观察及体外培养难度较大[4],因而很难对植原体病害的致病机理及病菌-寄主间互作机制进行深入探究。转录组学研究可以比较精准的反映寄主受到侵染后不同功能基因的响应机制,现已逐渐成为互作解析和差异基因分析的主要技术手段[5]。Fan和Mardi等人通过转录组分析发现,泡桐和墨西哥柠檬在被植原体侵染后,植原体-病菌互作基因表达上调,并且功能注释结果表明植株的相关代谢活动和信号通路主要与植物激素有关[6-7];万倩芸[8-9]等人在对茅苍术扁茎机制进行转录组分析时也发现扁茎症状可能与多种激素代谢通路协同作用相关,并且可能主要与病菌寄生后诱发植物代谢紊乱。而张舒仪等[10]通过对枣疯病植株进行转录组检测及qPCR后期验证后,认为植原体在侵染寄主后可能会迅速激起激素合成及相关保护酶合成基因的表达上升,进而导致激素含量和酶活性代谢紊乱,最终引发丛生、小叶等症状。前人结果表明转录组学与生理功能验证相结合可以较为准确、高效地揭示出植原体病害的致病机理,但目前关于国槐带化病内源激素与转录组学研究尚无报道。
本研究通过运用高效液相色谱对病健组织中的生长素、赤霉素、脱落酸、玉米素含量进行比较分析,并对感病和健康国槐枝条进行转录组测序和对比,结合GO和KEGG功能注释,筛选出可能导致国槐激素代谢紊乱的关键差异基因,旨在通过转录组学与生理水平相结合,揭示出国槐感病枝条扁茎机理。为国槐带化病的防治和抗病品种选育提供理论依据。
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通过对处于生长期的感病国槐(BZ)、健康国槐(JK)和感病国槐树上的表象健康枝(BJ)树皮的内源激素含量的测定,研究该病害发生期对国槐内源激素的影响。由图1可以看出,BZ、BJ和JK间的Zeatin、GA、IAA及ABA含量变化趋势基本一致。Zeatin含量在8、9月份达到高峰,之后呈下降趋势,其中8月份BZ中的Zeatin含量显著高于BJ和JK,BJ、JK无差异。其它月份中三者间无显著差异;GA含量与Zeatin含量年变化较为相似,在8月份达到最高,之后处于下降趋势。但其中8月份BZ的GA含量显著低于JK和BJ,BJ其次,BZ中的GA含量最低,其它月份三者间并无差异;而IAA和ABA的含量变化呈相反趋势,IAA除5月份最高外,之后一直呈下降趋势,并在11月份达到最低,其中JK含量最高,BJ其次,BZ中的IAA含量最低。ABA则是从5月份开始一直呈上升趋势,在11月份达到最高,BZ中的含量显著高于JK和BJ,BJ与JK的ABA含量在5、8、9月份中均无差异。
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通过对国槐感病样本(BZ1、BZ2、BZ3)和健康样本(JK1、JK2、JK3)进行高通量转录组测序,分别获得26499450、22444628、33026072、37221613、28464069和27856864条Raw Reads,在去除含有接头的Reads以及低质量的Reads(包括去除N的比例大于10%的Reads)后,最终得到20427416、17402101、25556038、28443464、21878012和21192357条Clean reads。其中Q20碱基百分比分别是98.20%、98.16%、98.05%、97.95%、97.93%和97.99%,GC碱基含量则均约达到44%(表1)。经过过滤后,最终得到可靠的转录本数据,并进行下一步生物学分析。
表 1 每个样品有效测序数据统计
Table 1. Statistics of valid sequencing data of each sample
SampleID Raw Reads Clean reads Q20/% GC/% BZ1 26499450 20427416 98.20 44.22 BZ2 22444628 17402101 98.16 44.47 BZ3 33026072 25556038 98.05 44.69 JK1 37221613 28443464 97.95 44.26 JK2 28464069 21878012 97.93 44.13 JK3 27856864 21192357 97.99 44.26 -
采用BLAST[14]软件对Unigene序列与NR、Swiss-Prot[15]、GO[16]、COG/KOG[17]、KEGG[18]数据库进行比对,共获得Unigne注释功能信息有72637条,占全部Unigene的60%,其中NR数据库中注释的信息最多,而COG数据库中被注释信息最少。通过使用KOBAS2.0[15]获得Unigene在KEGG中的KEGG Orthology结果,在预测Unigene的氨基酸序列之后使用HMMER[17]软件与Pfam[19]数据库比对,获得Unigene的注释信息(表2)。
表 2 Unigene功能注释统计
Table 2. Unigene function annotation statistics
Anno_Database Annotated_Number 300<=length<1000 length>=1000 COG 19618 5730 5909 GO 38153 17363 12172 KEGG 42076 19494 12612 KOG 35012 15753 10062 Pfam 32551 10775 13098 Swissprot 47071 19687 13780 nr 67685 31769 17211 All 72637 32877 17313 -
通过采用EBSeq[20]对所得数据进行差异分析,并采取Fold Change≥2且FDR<0.01作为筛选标准。如图2所示,绿点代表下调差异表达基因,红点代表上调差异表达基因,蓝色的点代表非差异表达基因。其中共得到差异表达基因数目4714条,国槐带化病样品相对于健康国槐样品上调基因有2283条,下调基因有2431条。这些结果表明,国槐在受到植原体侵染后其植物体内多数的基因表达受到抑制。
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通过对感病与健康国槐转录组差异表达基因进行GO功能注释富集分析,结果可分为3大类:细胞组成(cellular component)、分子功能(molecular function)、生物学过程(biological process)。其中注释到细胞组成有7个,分子功能10个,生物学过程3个(图3)。细胞组成中以膜的组成成分(783个)和质膜(141)中差异表达基因最多,另外膜的组成成分、运动蛋白复合物等离子组分以及细胞壁等差异表达基因富集表达均达到显著水平;分子功能中以蛋白丝氨酸苏氨酸激酶(136个)和DNA结合转录(84个)差异表达基因最多,而在木糖葡聚糖、木糖葡聚糖转化和DNA结合转录等富集基因中达到显著水平;生物学过程中蛋白磷酸化中的差异表达基因居多并达到显著水平。结果表明细胞组成和分子功能的基因富集更为显著,这说明在国槐受到植原体侵染以后,细胞和分子功能方面均发生明显的差异变化,并可能受到上述富集差异基因的调控。
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在生物体内,不同的基因产物相互协调来行使生物学功能,对差异表达基因的通路(Pathway)注释分析有助于进一步解读基因的功能。通过对差异基因(2539个)进行KEGG功能注释分析,发现差异基因被富集到48条代谢通路,本研究挑选了富集最显著的20条pathway条目在图中进行展示(图4)。其中代谢通路从低到高依次为:戊糖和葡萄糖醛酸互相转化、卟啉和叶绿素代谢、AMPK信号通路、癌变中的微型核糖核酸、多巴胺能受体、半光氨酸和蛋氨酸代谢、泛素介导的蛋白水解、MAPK信号通路、溶酶体、卵母细胞减数分裂、RNA降解、抗原加工和传递、苯丙氨酸合成、Ras信号通路、内噬作用、植物激素信号转导、植物-病原互作、淀粉和蔗糖代谢、Toll受体信号转导途径和NF-KB信号传导途径。其中,汇集差异基因最多的Toll受体信号转导途径和NF-KB信号传导途径均表现为显著下调,另外,控制细胞内噬作用、减数分裂以及抗原加工与传递同样表现为显著下调。
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感病国槐和正常国槐激素代谢通路的差异基因被注释到25个DEGs(图5)。由图可知,其中有7个下调基因(绿色标注),8个上调基因(红色标注)。其中,在色氨酸代谢通路上(生长素),AUX1节点上有3个差异表达基因下调,而在GH3节点有3个差异表达基因上调。而在玉米素信号传导代谢通路上(细胞分裂素),CRE1基因节点上涉及到7个差异基因下调;AHP节点上有3个差异基因下调;ARR-A节点处有3个差异基因下调。二萜生物合成(赤霉素)代谢通路上关键调控基因TF涉及13个差异基因上调。生长素、玉米素合成关键基因的下调以及赤霉素合成基因的上调,可能是导致国槐枝条顶端生长停滞、发生横向卷曲生长的主要原因。类胡萝卜素生物合成(脱落酸)代谢途径中,ABF调控关键基因涉及10个差异基因上调,产生这种原因可能与植原体的致病因子的基因干扰或植物体受到胁迫产生的自激反应有关。
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qRT-PCR验证本研究通过对转录组差异基因数据进行筛选获得与生长素、细胞分裂素、赤霉素和脱落酸代谢相关基因共6个(表3),其中,与生长素代谢相关基因AUX 1个(GHLAX5),与细胞分裂素信号传导受体CRE1相关基因3个(GHAHK4、GHAHP4和GHARR8),调控赤霉素信号传导的相关基因TF 1个(GHTF),脱落酸信号转导调控因子相关基因ABF 1个(GHABF2)。通过对上述6个激素代谢关键基因进行荧光定量表达验证,如图6所示,虽然定量表达与转录组结果具有一定差异,但表达趋势较为一致,生长素、细胞分裂素代谢关键基因表现为显著下调,赤霉素和脱落酸代谢关键基因表现为显著上调。
表 3 差异表达基因qRT-PCR 引物
Table 3. Primers of differentially expressed genes QRT-PCR
基因名称
Gene name序列 ID
Sequence ID上游引物
Forward sequence(5′→ 3′)下游引物
Reverse sequence(3′→ 5′)GHLAX5 DN122098_c0_g1 TCTCCCAACCTTCGAGAACC AATGCTAGGTGGGTGTGGAG GHAHK4 DN95401_c0_g1 CGCGGATGAAGAAAAGTGGTC CCAATTCCAGTGTCCTCCACA GHAHP4 DN115292_c1_g2 TGTTGGTCCGTTTGGGGATA AAGTGGAAGAGCAAGATTCCTACA GHARR8 DN107272_c1_g2 TGTTGGTCCGTTTGGGGATA AAGTGGAAGAGCAAGATTCCTACA GHTF DN111522_c0_g1 TCCCTTCCCATTTTTCCAGCA TTTTGTCTTGCTTCCACGCTC GHABF2 DN119624_c4_g2 GAGTTCTGAGGCCGTCCATT GAGCATACGCAATCTGCACT
感染国槐带化病植株内源激素及转录组分析
Analysis of Endogenous Hormones and Transcriptome in Sophora japonica Infected with Fasciation Disease
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摘要:
目的 为探究国槐带化病的致病机理,进而为国槐带化病的防治研究提供理论依据。 方法 以国槐健康与感病枝条作为研究材料,对其生长期间的5、8、9及11月份中枝条生长素、玉米素、赤霉素和脱落酸含量进行连续测定,通过选取8月份国槐的病、健组织进行转录组测序筛选差异表达基因,并进行qPCR验证分析。 结果 在5、8、9、11月中,健康、感病枝条及感病树上的健康枝条中的生长素、玉米素、赤霉素和脱落酸含量变化趋势基本一致,但在8月份国槐旺盛生长期间,感病枝条内的玉米素含量显著高于健康对照;生长素和赤霉素含量显著低于健康对照。而在11月份植株的生长后期,仅感病植株脱落酸含量显著高于健康对照。转录组测序结果表明差异基因下调表达明显高于上调,并且差异基因GO功能注释分析显示,差异基因主要汇集在细胞组分、分子功能和生物学过程3个方面,其中以细胞组分和分子功能差异基因富集更为显著;而KEGG代谢通路分析表明,感病植株的差异基因主要汇集在调控植株免疫应答的信号传导、植物-病原互作和植物激素信号传导等方面,并且其中调控植株免疫应答的信号传导富集差异基因最多且表现为显著下调;内源激素代谢通路注释结果表明,生长素、玉米素受体基因AUX1和CRE表达显著下调,而调控赤霉素、脱落酸代谢的基因TF和ABF表达显著上调;通过对转录组数据进行筛选获得调节生长素、玉米素、赤霉素和脱落酸代谢通路代谢及信号转导的相关基因(GHLAX5、GHAHK4、GHAHP4、GHARR8、GHTF和GHABF2)进行qPCR验证,结果虽与转录组数据存在一定差异,但表达趋势基本一致,这些结果表明,上述基因的差异表达可能对于调控国槐内源激素含量变化具有重要作用。 结论 认为玉米素含量的显著上升和生长素、赤霉素含量的显著降低,阻碍植株的免疫机制应答并调控这3种激素代谢的关键基因差异表达,可能是导致国槐带化病枝条发病且横向生长形成扁茎的主要原因。 Abstract:Objective To investigate the pathogenesis of banding disease of Sophora japonica, so as to provide data for the prevention and control of the disease. Method In this study, the contents of auxin, zeatin, gibberellin and abscisic acid in the healthy and diseased branches of Sophora japonica were determined continuously in May, August, September and November. The differentially expressed genes were screened by transcriptome sequencing and the qPCR analysis was performed. Result In May, August, September and November, the contents of auxin, zeatin, gibberellin and abscisic acid in healthy tree, infected and symptomless shoots of the diseased trees were basically the same, but during the vigorous growth period of S. japonica in August, the content of zeatin in the infected branches was significantly higher than that in the healthy control; the contents of auxin and gibberellin were significantly lower than that of the healthy control. However, in the late growth stage of the plants in November, only the abscisic acid content of the diseased plants was significantly higher than that of the healthy control. Transcriptome sequencing results showed that the down-regulation of differential genes was significantly higher than up-regulation, and GO functional annotation analysis of differential genes showed that the differential genes were mainly concentrated in cell component, molecular function and biological process, especially in cell component and molecular function, while the KEGG metabolic pathway analysis showed that the differentially expressed genes in susceptible plants were mainly concentrated in signal transduction, plant pathogen interaction and plant hormone signal transduction. The results of endogenous hormone metabolic pathway annotation showed that AUX1 and CRE were down regulated, while the TF and ABF were up-regulated. The genes (GHLAX5, GHAHK4, GHAHP4,GHARR8, GHTF and GHABF2) regulating auxin, zeatin, gibberellin and abscisic acid metabolism and signal transduction were obtained by screening the transcriptome data for qPCR verification. Although there were some differences between the results and the transcriptome data, the expression trend was basically the same. These results suggested that the differential expression of these genes may play an important role in regulating the endogenous hormone content of S. japonica. Conclusion The significant increase of zeatin content and the significant decrease of auxin and gibberellin contents may hinder the immune response of plants and the differential expression of key genes regulating the metabolism of these three hormones, which may be the main reason for the occurrence and lateral growth of the diseased branches of S. japonica. -
Key words:
- Sophora japonica
- / fasciation disease
- / pathogenesis
- / endogenous hormones
- / transcriptome
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表 1 每个样品有效测序数据统计
Table 1. Statistics of valid sequencing data of each sample
SampleID Raw Reads Clean reads Q20/% GC/% BZ1 26499450 20427416 98.20 44.22 BZ2 22444628 17402101 98.16 44.47 BZ3 33026072 25556038 98.05 44.69 JK1 37221613 28443464 97.95 44.26 JK2 28464069 21878012 97.93 44.13 JK3 27856864 21192357 97.99 44.26 表 2 Unigene功能注释统计
Table 2. Unigene function annotation statistics
Anno_Database Annotated_Number 300<=length<1000 length>=1000 COG 19618 5730 5909 GO 38153 17363 12172 KEGG 42076 19494 12612 KOG 35012 15753 10062 Pfam 32551 10775 13098 Swissprot 47071 19687 13780 nr 67685 31769 17211 All 72637 32877 17313 表 3 差异表达基因qRT-PCR 引物
Table 3. Primers of differentially expressed genes QRT-PCR
基因名称
Gene name序列 ID
Sequence ID上游引物
Forward sequence(5′→ 3′)下游引物
Reverse sequence(3′→ 5′)GHLAX5 DN122098_c0_g1 TCTCCCAACCTTCGAGAACC AATGCTAGGTGGGTGTGGAG GHAHK4 DN95401_c0_g1 CGCGGATGAAGAAAAGTGGTC CCAATTCCAGTGTCCTCCACA GHAHP4 DN115292_c1_g2 TGTTGGTCCGTTTGGGGATA AAGTGGAAGAGCAAGATTCCTACA GHARR8 DN107272_c1_g2 TGTTGGTCCGTTTGGGGATA AAGTGGAAGAGCAAGATTCCTACA GHTF DN111522_c0_g1 TCCCTTCCCATTTTTCCAGCA TTTTGTCTTGCTTCCACGCTC GHABF2 DN119624_c4_g2 GAGTTCTGAGGCCGTCCATT GAGCATACGCAATCTGCACT -
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