Cloning and expression analysis of chalcone synthase gene from Koelreuteria paniculata
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摘要:目的 查尔酮合酶(CHS)是苯丙烷途径的限速酶之一,在植物次生代谢物的合成中起着重要的作用。本研究通过对栾树CHS 基因进行克隆与生物信息学分析,以及分析栾树 CHS 基因表达与类黄酮合成的关系,期望为后续深入研究栾树类黄酮代谢途径其他相关基因、 CHS 基因家族以及锦叶栾呈色机制提供参考。方法 以栾树叶片为材料,采用RT-PCR技术进行查尔酮合酶基因的克隆并进行生物信息学分析;通过实时定量PCR(qRT-PCR)技术分析 CHS 基因在栾树不同组织以及在5月、7月、9月的栾树和锦叶栾叶片中的表达模式;通过代谢组测定筛选出栾树与锦叶栾的类黄酮差异代谢物。结果 克隆获得两个 CHS 基因的全长DNA,命名为 KpCHS1 和 KpCHS2 。其中 KpCHS1 序列全长为2 492 bp,ORF为1 173 bp,编码含有390个氨基酸的蛋白质; KpCHS2 序列全长为1 321 bp,ORF为1 182 bp,编码含有393个氨基酸的蛋白质;进一步的序列比对和系统发育分析表明,KpCHS1和KpCHS2蛋白高度同源,具有四个CHS特异性保守基序和一个查尔酮合成酶活性位点; KpCHS1 和 KpCHS2 在栾树根、茎、叶、种子中都普遍表达,其中, KpCHS2 在种子中的表达量最高, KpCHS1 在叶片中表达量高,而在根和茎中,两个基因的表达量相似且较低;表达模式分析显示,在栾树和锦叶栾叶片中,随着月份增加, KpCHS1 的表达量呈现出下降的趋势,而 KpCHS2 表达量未表现出明显的规律。在7月份的叶片样本中, KpCHS1 基因在锦叶栾中表达量显著高于栾树;代谢组结果显示,山奈酚-7-O-葡萄糖苷、7-羟基香豆素、槲皮素-3β-D-葡萄糖苷、以及类黄酮生物合成途径重要的中间产物山萘酚、柚皮苷等黄酮类物质在锦叶栾叶中含量显著升高。结论 KpCHS1和KpCHS2属于栾树查尔酮合酶家族并且高度同源,但在系统进化树上分布在很远分支上,推测这两个蛋白在氨基酸活性催化功能上可能存在较大差异;KpCHS1和KpCHS2在根、茎、叶、种子中均有表达,且在叶和种子中较高。研究结果初步显示,KpCHS1基因与栾树类黄酮的生物合成高度相关。Abstract:Objective Chalcone synthase (CHS) is one of the rate-limiting enzymes of phenylpropanoid pathway which plays superior roles in the production of secondary metabolites. In this study, by cloning and bioinformatics analysis of CHS gene and analyzing the relationship between CHS gene expression and flavonoid synthesis of Koelreuteria paniculata, we hope to provide reference for further study of flavonoid biosynthesis pathway related genes, evolution of CHS gene family and coloration mechanism of Koelreuteria paniculata ‘Jinye’.Method CHS genes were isolated and characterized by RT-PCR from Koelreuteria paniculata. And the expression patterns of CHS gene in different tissues of Koelreuteria paniculata and in the leaves of Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’
in May, July and September were analyzed by qRT-PCR; the differential flavonoid metabolism between Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’ was screeidues ether. Result Two full-length DNA of CHS genes were cloned named KpCHS1 and KpCHS2 . The KpCHS1 gene sequence was found to be 2 492 bp and comprised an open reading frame of 1 173 bp, encoding for 390 amino acid residues, the KpCHS2 gene sequence was found to be 1 321 bp and comprised an open reading frame of 1 182 bp, encoding for 393 amino acid residues ether. Alignment of the predicted amino acid sequence of KpCHS2 had been shown to have high sequence similarity with KpCHS1, with four CHS specific conserved motifs and one chalcone synthase active site. Furthermore, KpCHS1 and KpCHS2 were generally expressed in roots, stems, leaves and seeds of Koelreuteria paniculata. Among them, the expression of KpCHS2 was the highest in seeds, while that of KpCHS1 was higher in leaves. In roots and stems, the expression levels of the two genes were similar and lower. The expression pattern analysis showed that in Koelreuteria paniculata and Koelreuteria paniculata ‘ Jinye’, the expression of KpCHS1 decreased with the increase of months, while the expression of KpCHS2 did not show obvious regularity. In the July plant samples, the expression of KpCHS1 gene in Koelreuteria paniculata ‘Jinye’ was higher than that in Koelreuteria paniculata. Besides, we analyzed the metabonomics of Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’ leaves in July, and screened out the different flavonoids. It was found that kaempferol-7-o-glucoside, 7-hydroxycoumarin, quercetin-3β-D-glucoside, and kaempferol, naringin, which were important intermediate products in flavonoid biosynthesis, were significantly increased in Koelreuteria paniculata ‘Jinye’ leaves. Conclusion KpCHS1 and KpCHS2 belong to the chalcone synthase family of Koelreuteria paniculata and are highly homologous, but they are distributed in far branches of the phylogenetic tree. It is speculated that the two proteins may have great differences in the catalytic function of amino acid activity. KpCHS1 and KpCHS2 are expressed in roots, stems, leaves and seeds, and higher in leaves and seeds of Koelreuteria paniculata. Our results indicate that the expression of KpCHS1 gene is highly related to the synthesis of flavonoids in Koelreuteria paniculata.-
Keywords:
- Koelreuteria paniculata /
- chalcone synthase /
- cloning /
- flavonoid biosynthesis
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栾树(Koelreuteria paniculata)属于无患子科(Sapindaceae)栾树属植物,是我国特有的乡土树种,具有适应性强、对自然灾害抗逆性强[1]等优点,作为行道树在我国广泛种植。栾树具有生态保护[2]、城乡美化[3]的作用,由于富含黄酮类物质,使得其成为兼具药用价值[4]的优良树种。锦叶栾(K. paniculata ‘Jinye’)是由普通栾树芽变选育而来的一种彩叶(金黄色)突变体[5],其生理学特征与普通栾树类似,但叶色发生明显变异。每年的5月,锦叶栾新生出金黄色叶片,嫩茎、嫩梢呈鲜艳的橙红色。但从7月至9月,锦叶栾的叶色发生复绿的现象,即由金黄转变成黄绿色。先前的研究表明[6],在其旺盛生长阶段(5—6月),锦叶栾叶片具有较高的类黄酮含量,且在5月底时达到峰值(36.72 mg/g),之后便一直呈现下降趋势,9月时含量已降至20 mg/g,锦叶栾叶片内总黄酮含量的季节性变化规律与其叶色变化规律一致。研究表明[7],高等植物叶色主要取决于叶绿素、类胡萝卜素、类黄酮这几种色素的含量和比例,类黄酮含量的变化可能是锦叶栾叶色变异的原因之一。
类黄酮(flavonoids)是一大类植物次生代谢物,包括7 000多种化合物[8],基本结构是C6-C3-C6。其在植物体内广泛分布于花、果、叶中,并表现出多样化的生物功能,如植物防御、抗氧化、参与信号传导、防护屏蔽紫外线以及作为结构支持等[9-10]。植物中类黄酮生物合成途径已经被科学家们研究的较为透彻[11-12]。查尔酮合酶(chalcone synthase, CHS)是类黄酮合成途径中的第一个限速酶,其催化一分子的4-香豆酰CoA和三分子丙二酰CoA合成查尔酮,再经查尔酮异构酶(CHI)快速的空间异构反应异化成无色的柚皮素,柚皮素作为主要的中间体物质进入下游3个类黄酮代谢阶段:花青素和原花青素的合成、黄酮和异黄酮的合成以及黄酮醇的合成[13]。因此,了解查尔酮合酶的功能基因及其调控机制对探索类黄酮和花色素的生物合成途径的遗传调控至关重要。CHS基因在类黄酮合成中的重要作用已经在多种植物中得到了验证[14-17],但迄今为止尚未成功克隆出栾树CHS基因。为了解CHS基因在栾树类黄酮生物合成中的作用,本研究利用生物信息学方法从栾树转录组中检索并克隆得到2个CHS基因,并对其蛋白质理化性质、进化关系、保守功能域以及表达模式进行了分析,为后续深入研究类黄酮代谢途径相关基因、CHS基因表达调控以及CHS基因家族进化提供科学依据。
1. 材料与方法
1.1 材 料
栾树与锦叶栾在山西省襄垣县栾树苗圃自然条件下生长。我们选取两个品种各3株长势一致的成年植株,收集种子,并于2018年5月12日、7月12日和9月12日在其分枝顶部同样位置采集叶片组织。同时取北京林业大学实验楼组培间内栾树组培苗的根与茎作为试验材料。各样品经液氮速冻后保存于−80 ℃冰箱中。
1.2 基因克隆
1.2.1 RNA提取与cDNA合成
利用植物总RNA提取试剂盒(OMEGA,美国)提取栾树根、茎、叶、种子和锦叶栾叶片的总RNA,进行RNA质量及浓度检测后,使用反转录试剂盒(Promega,美国)合成cDNA第一条链,−20 ℃保存备用。
1.2.2 KpCHS基因的克隆
根据本课题组的栾树转录组数据,利用Primer3Plus在线网站设计克隆特异性引物(见表1)。以栾树和锦叶栾cDNA为模板,进行PCR扩增获得目的片段。利用DNA凝胶回收试剂盒(Biomega,中国)回收目的条带,将其与pGEM-T载体连接,转化至大肠杆菌top10感受态中,涂板过夜培养。挑单菌落进行PCR验证,将鉴定为阳性结果的单菌落加入到LB液体培养基中,放于37 ℃恒温摇床培养,将培养好的菌液送往睿博生物公司进行测序。
表 1 基因克隆中使用引物列表Table 1. List of primers in gene cloning引物名称 Primer name 序列(5′—3′) Sequence (5′−3′) Tm值 Tm value 用途 Usage CHS1-F ATGGTGACTGTCGAGGAAGTC 55.00 KpCHS1 基因扩增 KpCHS1 gene amplification CHS1-R CTAAGCAGAGGCAACAGAGTGG 55.00 KpCHS1 基因扩增 KpCHS1 gene amplification CHS2-F ATCTCACTCCTAAACCCCCTTC 56.00 KpCHS2 基因扩增 KpCHS2 gene amplification
CHS2-R TTTAATAAAAGGAACAGTATCCAGA 56.00 KpCHS2 基因扩增 KpCHS2 gene amplification Actin-F AAATTAACGAGGACACCAATGC 58.00 qRT-PCR Actin-R GGGTATGGATATGGCGATCTTA 58.00 qRT-PCR Y-CHS1-F GTGTCGAAAAGCCCATGTATGA 58.99 qRT-PCR Y-CHS1-R TTGAAATCAGCCCAGGAACATC 58.91 qRT-PCR Y-CHS2-F AAGAACATCGAGAAAAGCTTGG 59.89 qRT-PCR Y-CHS2-R GCCCTGAGTTTCTCTTCTTTGA 60.00 qRT-PCR 1.2.3 生物信息学分析
采用DNAMAN软件推导其氨基酸序列,并分析氨基酸组成成分和理化性质;利用NCBI数据库(https://www.ncbi.nlm.nih.gov/)中的Blastp工具获得序列相似性较高的几个物种氨基酸序列;采用TMpred在线软件(https://embnet.vital-it.ch/software/TMPRED_form.html)分析蛋白质跨膜区域;利用PSORT在线软件(http://psort.hgc.jp/)预测蛋白质亚细胞定位;采用NCBI数据库中的CDD数据库对蛋白质的特异结构域进行分析;使用SOMPA在线网站(http://bip.weizmann.ac.il/bio-tools/faq.html)对KpCHS蛋白进行二级结构预测;通过使用Swiss-model和WebLab ViewerLite(http://www.accelrys.com)用于蛋白三级结构预测。采用PROSITE在线分析(https://prosite.expasy.org/)工具分析蛋白的活性位点,最后利用MEGA 7.0软件[18]进行系统进化树的构建。
1.2.4 组织表达特异性分析与不同品种叶片表达量分析
根据测序结果,设计实时荧光定量引物,以Actin作为内参基因,对其在栾树根、茎、叶、种子4个组织中的表达量进行分析。同时,以栾树与锦叶栾叶片cDNA为模版,检测CHS基因在两个品种不同月份的叶片中具体表达情况。利用GraphPad软件[19]进行显著性分析与柱状图的绘制。
1.3 黄酮类代谢物测定
1.3.1 代谢物提取
选取7月份的栾树、锦叶栾叶片经液氮研磨后分别称取100 mg,粉末转入1.5 mL EP管中,加入预冷的400 μL甲醇/水(3∶1),涡旋混合,于4 ℃下放置过夜,之后13000 rpm,4 ℃离心15 min,取上清经过0.22 μm滤膜过滤后,氮气吹干,−80 ℃保存待用,进行LC-MS质谱检测前,用异丙醇/甲醇/水(1∶1∶2 )50 μL复溶后,离心取上清进行检测。
1.3.2 测定与分析
整个分析过程中样品置于4 ℃自动进样器中,样品使用C18色谱柱进行分离。其中进样量3 μL,柱温45 ℃,流速0.35 mL/min;色谱流动相A:0.1%甲酸水溶液,B:乙腈与0.1%甲酸混合液;色谱梯度洗脱程序如下:0 ~ 0.5 min,98% A;0.5 ~ 15 min,98% ~ 2% A;15 ~ 17 min,2% A;17 ~ 20 min,2% ~ 98% A。样品经UPLC分离后用Q-Exactive质谱仪(Thermo Fisher)进行质谱分析。之后利用SIMCA-P软件对数据进行处理。进行可信度检验后,以FC(fold change)分析(FC > 2或FC < 0.5和P值 < 0.05作为筛选标准)和VIP(variable importance for the Projection)(VIP > 1和P值 < 0.1作为筛选标准)筛选出黄酮类差异代谢物。
2. 结果与分析
2.1 CHS基因的克隆与氨基酸序列分析
以栾树叶片的DNA和cDNA为模板,设计特异性引物,成功克隆出两个CHS基因,命名为KpCHS1
和KpCHS2(MW556008,MW556009)(图1)。利用GSDS 2.0[20]在线分析工具对两个基因结构进行分析,得到基因外显子与内含子分布情况如图2所示,KpCHS1和KpCHS2都有两个外显子和一个内含子,KpCHS1外显子长度分别为178 bp和995 bp,内含子长度为1 319 bp;KpCHS2外显子长度分别为180 bp和1 002 bp,内含子长度为139 bp。利用DNAMAN软件对测序结果进行序列分析,结果显示KpCHS1基因包含完整的cDNA开放阅读框(ORF),ORF全长1 173 bp,编码含有390个氨基酸的蛋白质,KpCHS2基因序列全长1 182 bp,编码含有393个氨基酸的蛋白质。由表2可见,KpCHS1和KpCHS2都为疏水性蛋白,不具有跨膜区域。蛋白质亚细胞定位预测结果显示两个蛋白都定位在细胞质中。 图 1 KpCHS1和KpCHS2基因组全长及CDS序列 M. DL3000 Marker;1、2、3、4分别代表KpCHS1全长基因、KpCHS1 CDS序列、KpCHS2全长基因、KpCHS2 CDS序列。M, DL3000 Marker; 1, 2, 3, 4 represent KpCHS1 full-length gene, KpCHS1 CDS sequence, KpCHS2 full-length gene and KpCHS2 CDS sequence, respectively.Figure 1. Full length and CDS sequence of KpCHS1 and KpCHS2 genes表 2 CHS蛋白理化性质Table 2. Physicochemical properties of CHS protein蛋白名称
Protein name氨基酸数量
Quantity of
amino acids分子量
Molecular mass/kD等电点
Isoelectric point (pI)亲/疏水性
Hydrophilicity/
hydrophobicity跨膜区域
Transmembrane
region亚细胞定位预测
Prediction of protein
subcellular localizationKpCHS1 390 42.59 6.12 疏水性 Hydrophobicity 不具有 No 细胞质 Cytoplasm KpCHS2 393 42.91 6.57 疏水性 Hydrophobicity 不具有 No 细胞质 Cytoplasm 2.2 栾树CHS蛋白的二级与三级结构分析
二级结构预测分析表明,KpCHS1由45.13%的α螺旋,15.13%的延伸链,7.18%的β转角和32.56%的无规则卷曲组成。KpCHS2由43.00%的α螺旋,16.03%的延伸链,6.87%的β转角和34.10%的无规则卷曲组成。α螺旋和无规则卷曲构成了二级结构主要部分的交错控制(图3)。通过Swiss-Modeling分析了基于同源性的KpCHS蛋白的3D结构建模,通过分析得到的模型,发现栾树CHS蛋白三级结构大部分由α螺旋和不规则卷曲组成,与草本植物异叶天南星(Arisaema heterophyllum)[21]、木本植物血橙(Citrus sinensis)[22]等CHS蛋白结构相似度很高,从蛋白的水平上证明查尔酮合酶较为保守(图4)。
2.3 栾树CHS蛋白同源序列及系统进化分析
将测序结果利用DNAMAN软件翻译获得CHS氨基酸序列,将KpCHS1和KpCHS2氨基酸序列进行blastp搜索,结果显示KpCHS1与鸡爪槭CHS(Acer palmatum, AWN08245.1)同源性最高,覆盖率达99%,匹配度达94.87%。KpCHS2与荔枝CHS(Litchi chinensis,ADB44077.1)同源性最高,覆盖率达100%,匹配度达95.93%。从栾树中克隆获得的2个CHS几乎都具有CHS模型结构的所有主要特征,说明KpCHS1和KpCHS2属于查尔酮合成酶家族。它们在整个编码区具有高度相似性,有4个CHS特异性保守基序(标记为Ⅰ、Ⅱ、Ⅲ和Ⅳ)和29个保守氨基酸残基,可分为4类[23](图5)。利用PROSITE在线网站分析栾树CHS蛋白功能位点,发现该蛋白具有一个查尔酮合成酶活性位点(RLMMYQQGCFAGGTVLR)。用MEGA7.0软件中自带的Cluster W进行蛋白序列多重比对后,用邻接法(neighbor-joining)以1000个重复进行Bootstrap检验,绘制出包括KpCHS1和KpCHS2等17个CHS蛋白家族的进化树(图6)。根据系统进化树分析,KpCHS1与鸡爪槭ApCHS、柠檬(Citrus limon, AXJ20608.1)ClCHS、柑橘(Citrus clementina, XP 006420608.1)CcCHS等关系较近聚为一类,而与KpCHS2在聚类分支上距离较远,说明两个基因在氨基酸活性催化功能上存在一定的差异性。
图 5 KpCHS1和KpCHS2编码的氨基酸同源比对在所有序列中高度保守的残基以黑色背景表示,在序列中仅部分保守的残基以白色和灰色背景显示。蛋白活性位点、催化残基、CoA结合位点、其他高度保守的残基和不同的残基分别表示为◆、△、●、□和♡。红框为查尔酮合成酶活性位点(RLMMYQQGCFAGGTVLR)。Highly conserved residues in all sequences are indicated in black background and only partially conserved residues in the CHS sequences are showed in black with gray background. The protein sequences of KpCHS have four CHS-specific conserved motifs (marked Motif Ⅰ, Ⅱ, Ⅲ and Ⅳ). Active site, catalytic residues, CoA-binding site, other highly conserved residues and different residues are indicated as ◆, △, ●, □ and ♡, respectively. The red box is the active site of chalcone synthase (RLMMYQQGCFAGGTVLR).Figure 5. Amino acid sequence alignment encoded by KpCHS1 and KpCHS22.4 CHS基因表达模式分析
我们根据测序结果设计荧光定量引物(表1),利用qRT-PCR检测了栾树中KpCHS1
和KpCHS2的组织特异性以及在不同品种时期的叶片中的表达模式。结果显示,虽然KpCHS1和KpCHS2在本研究的所有组织中都普遍表达,但他们的相对表达量却有所不同,与其他组织相比,KpCHS2在种子中的表达量最高,KpCHS1在叶片中表达量最高,而在根和茎中,两个基因的表达量相似且较低(图7a)。同时,我们利用荧光定量探究KpCHS1(图7b)和KpCHS2(图7c)在5、7、9三个月份的的栾树和锦叶栾叶片中的表达模式。结果显示,随着月份增加,KpCHS1的表达量呈现出下降的趋势,而KpCHS2表达未表现出明显的规律。除了7月份,KpCHS1基因在锦叶栾中表达量高于栾树,其余月份中,KpCHS1 和KpCHS2在栾树中的表达量都高于锦叶栾。 图 7 栾树不同器官以及不同月份栾树和锦叶栾叶片中KpCHS1和KpCHS2基因表达分析 a. KpCHS1和KpCHS2的组织特异性表达;b. KpCHS1在WT和GL的5月、7月、9月叶片中的表达量分析;c. KpCHS2在WT和GL的5月、7月、9月叶片中的表达量分析;WT为栾树;GL为锦叶栾。a, tissue specific expression of KpCHS1 and KpCHS2; b, expression of KpCHS1 in the leaves of WT and GL in May, July and September; c, expression of KpCHS2 in the leaves of WT and GL in May, July and September; WT, Koelreuteria paniculata; GL, K. paniculata ‘Jinye’.Figure 7. Analysis of KpCHS1 and KpCHS2 expression in different organs and leaves of Koelreuteria paniculata and K. paniculata ‘Jinye’ in different months2.5 黄酮类代谢物测定
查尔酮合酶是类黄酮代谢途径的第一个限速酶,它的表达会影响下游类黄酮和花色素苷的积累。为了探究栾树和锦叶栾叶片中的类黄酮物质积累情况,我们使用LC-MS技术分析了7月份锦叶栾和栾树叶片之间的代谢物水平,筛选出差异的类黄酮代谢物,并利用tbtools软件[24]进行热图的制作。如图8所示,可以看出山奈酚-7-O-葡萄糖苷、7-羟基香豆素、槲皮素-3β-D-葡萄糖苷、以及类黄酮生物合成中重要的中间产物山萘酚、柚皮苷等黄酮类物质在锦叶栾叶中含量显著升高。
3. 讨 论
自1972年Kreuzaler[25]等在荷兰芹(Petroselinum crispum)悬浮细胞中首次提取出查尔酮合酶,开启了科学家们对CHS基因的研究。这些年来,近百种植物的CHS家族基因被陆续报道[26-28]。研究表明,查尔酮合成酶基因保守性很强,编码区长约1 200 bp,编码约400个氨基酸,除了已发现的金鱼草(Antirrhinum majus)的AMCHS含有两个内含子之外,其余CHS基因均只包括两个外显子和一个内含子,内含子长度从几十到几千碱基不等[28]。本研究克隆得到KpCHS1序列全长为2 492 bp,CDS区为1 173 bp,编码含有390个氨基酸的蛋白质;KpCHS2序列全长为1 321 bp,CDS区为1 182 bp,编码含有393个氨基酸的蛋白质。研究表明,种属间的CHS蛋白序列相似性在74% ~ 98%之间,大多数同科同属甚至同物种的CHS分布在很近的分支上,少数分布在较远的分支中。从系统进化树上来看,KpCHS1和KpCHS2分布较远,属于少数的分布在很远分支上的基因,推测这两个蛋白在氨基酸活性催化功能上可能存在较大的差异[29]。
利用qRT-PCR技术分析了KpCHS1和KpCHS2在栾树不同组织中的表达模式,同时结合代谢组数据,研究了栾树和锦叶栾不同时期叶片中的CHS基因表达情况。结果显示,KpCHS1和KpCHS2在栾树的多种植物器官中以不同水平积累(图4),但在叶片和种子中都呈高水平表达,推测可能与其含有丰富的黄酮类物质有关,先前研究结果显示栾树叶片、种子的粗提物中含有丰富的次生代谢产物,如没食子酸衍生物、氰基类化合物和类黄酮化合物等,具有潜在的药用和抗菌性能。本项研究中,5月至9月,栾树与锦叶栾叶片中KpCHS1基因表达量整体呈下降趋势,与前人研究的锦叶栾叶片中类黄酮物质含量季节性下降趋势一致[6],同时,代谢组测定结果显示,相较于栾树,7月份的锦叶栾叶片中黄酮类物质含量发生显著性升高,与KpCHS1在锦叶栾中的高表达结果一致(图7),说明KpCHS1基因与栾树类黄酮的生物合成高度相关。在高等植物中,CHS基因通常以3 ~ 12个成员的多基因家族形式存在[30],例如苹果(Malus domestica)有3个成员[31],桑树(Morus alba)有5个[32]。CHS基因的不同成员在表达模式、表达时间上不尽相同,在不同器官、不同品种方面也存在表达差异[33],与KpCHS2基因和KpCHS1基因在各种植物组织中不同的表达模式结果一致。
CHS基因的表达受多种因素影响,光照[34]、环境胁迫[35]、生物胁迫如病原菌、虫害[36]以及某些中间产物都能影响CHS的生物活性。高光照强度[37]和UV-B辐射[38]在类黄酮生物合成的调控中也起着重要作用。一般植物例如丁香(Syringa oblata)[39]和槐果(Sophora japonica)[40]响应光照强度主要表现为在5月至10月间,类黄酮等物质在8月达到峰值然后逐渐下降。而大多数叶色突变体常伴随着叶绿体结构发育缺陷[41-42],则需要调整其生长发育以适应各种环境条件,包括光信号[43]、高温和遮荫能力[44]。Song等[45]通过对“黄金芽”突变体茶树进行中度遮阴处理模拟光照强度下降,研究发现在光照强度下降的条件下CHS基因表达与类黄酮物质含量显著降低。推测锦叶栾中类黄酮物质在5月到9月的下降趋势可能是由于对夏季高温强光的响应调节。虽然植物叶色突变体大多为单基因突变[46],但往往会导致复杂的转录和代谢水平的变化,而植物次生代谢调控又是极其复杂的过程,对于栾树叶色突变体研究还需进一步的多代谢途径及多时间点的动态研究。本研究中,我们期望通过对栾树CHS基因进行克隆与分析,为进一步研究栾树类黄酮化合物的合成及其调控机制提供科学依据,为深入揭示锦叶栾叶片呈色机理提供理论参考。
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图 1 KpCHS1
和KpCHS2基因组全长及CDS序列 M. DL3000 Marker;1、2、3、4分别代表KpCHS1全长基因、KpCHS1 CDS序列、KpCHS2全长基因、KpCHS2 CDS序列。M, DL3000 Marker; 1, 2, 3, 4 represent KpCHS1 full-length gene, KpCHS1 CDS sequence, KpCHS2 full-length gene and KpCHS2 CDS sequence, respectively.
Figure 1. Full length and CDS sequence of KpCHS1 and KpCHS2 genes
图 5 KpCHS1和KpCHS2编码的氨基酸同源比对
在所有序列中高度保守的残基以黑色背景表示,在序列中仅部分保守的残基以白色和灰色背景显示。蛋白活性位点、催化残基、CoA结合位点、其他高度保守的残基和不同的残基分别表示为◆、△、●、□和♡。红框为查尔酮合成酶活性位点(RLMMYQQGCFAGGTVLR)。Highly conserved residues in all sequences are indicated in black background and only partially conserved residues in the CHS sequences are showed in black with gray background. The protein sequences of KpCHS have four CHS-specific conserved motifs (marked Motif Ⅰ, Ⅱ, Ⅲ and Ⅳ). Active site, catalytic residues, CoA-binding site, other highly conserved residues and different residues are indicated as ◆, △, ●, □ and ♡, respectively. The red box is the active site of chalcone synthase (RLMMYQQGCFAGGTVLR).
Figure 5. Amino acid sequence alignment encoded by KpCHS1 and KpCHS2
图 7 栾树不同器官以及不同月份栾树和锦叶栾叶片中KpCHS1
和KpCHS2基因表达分析 a. KpCHS1和KpCHS2的组织特异性表达;b. KpCHS1在WT和GL的5月、7月、9月叶片中的表达量分析;c. KpCHS2在WT和GL的5月、7月、9月叶片中的表达量分析;WT为栾树;GL为锦叶栾。a, tissue specific expression of KpCHS1 and KpCHS2; b, expression of KpCHS1 in the leaves of WT and GL in May, July and September; c, expression of KpCHS2 in the leaves of WT and GL in May, July and September; WT, Koelreuteria paniculata; GL, K. paniculata ‘Jinye’.
Figure 7. Analysis of KpCHS1 and KpCHS2 expression in different organs and leaves of Koelreuteria paniculata and K. paniculata ‘Jinye’ in different months
表 1 基因克隆中使用引物列表
Table 1 List of primers in gene cloning
引物名称 Primer name 序列(5′—3′) Sequence (5′−3′) Tm值 Tm value 用途 Usage CHS1-F ATGGTGACTGTCGAGGAAGTC 55.00 KpCHS1 基因扩增 KpCHS1 gene amplification CHS1-R CTAAGCAGAGGCAACAGAGTGG 55.00 KpCHS1 基因扩增 KpCHS1 gene amplification CHS2-F ATCTCACTCCTAAACCCCCTTC 56.00 KpCHS2 基因扩增 KpCHS2 gene amplification
CHS2-R TTTAATAAAAGGAACAGTATCCAGA 56.00 KpCHS2 基因扩增 KpCHS2 gene amplification Actin-F AAATTAACGAGGACACCAATGC 58.00 qRT-PCR Actin-R GGGTATGGATATGGCGATCTTA 58.00 qRT-PCR Y-CHS1-F GTGTCGAAAAGCCCATGTATGA 58.99 qRT-PCR Y-CHS1-R TTGAAATCAGCCCAGGAACATC 58.91 qRT-PCR Y-CHS2-F AAGAACATCGAGAAAAGCTTGG 59.89 qRT-PCR Y-CHS2-R GCCCTGAGTTTCTCTTCTTTGA 60.00 qRT-PCR 表 2 CHS蛋白理化性质
Table 2 Physicochemical properties of CHS protein
蛋白名称
Protein name氨基酸数量
Quantity of
amino acids分子量
Molecular mass/kD等电点
Isoelectric point (pI)亲/疏水性
Hydrophilicity/
hydrophobicity跨膜区域
Transmembrane
region亚细胞定位预测
Prediction of protein
subcellular localizationKpCHS1 390 42.59 6.12 疏水性 Hydrophobicity 不具有 No 细胞质 Cytoplasm KpCHS2 393 42.91 6.57 疏水性 Hydrophobicity 不具有 No 细胞质 Cytoplasm -
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