玉米pTAC2影响苗期叶片叶绿素合成的转录组分析

doi:10.3864/j.issn.0578-1752.2020.05.002

摘要/Abstract

摘要：

【目的】叶绿素是参与光合途径最为重要的光合色素。叶绿体的发育及叶绿素的合成在很大程度上依赖于质体基因组与核基因组之间的双向信号传导来精确协调基因表达。通过对白化表型的CRISPR/Cas9-ZmpTAC2转基因阳性纯合突变材料进行RNA-seq研究,筛选和鉴定参与叶绿素合成的相关基因,为明确叶绿素的合成途径奠定基础。【方法】以CRISPR/Cas9-ZmpTAC2玉米转基因编辑纯合突变株系为研究材料,使用透射电镜观察叶绿体超微结构和分光光度法测定叶片叶绿素含量,确定叶绿体发育状态及叶绿素合成情况。对转基因阴性材料（CK）和CRISPR/Cas9-ZmpTAC2转基因纯合编辑材料（zmptac2）苗期叶片取样进行转录组测序。通过生物信息学分析,寻找CK与zmptac2间差异表达的基因;qRT-PCR对差异表达基因进行验证。通过酵母双杂交筛选与玉米pTAC2互作的蛋白质。【结果】共获得15株T₀转基因植株,包括绿色植株（7株）和白色植株（8株）。绿色幼苗中3株为转基因阴性材料,4株为转基因阳性（2株为未编辑,2株为杂合编辑突变）,白色植株(8株)均为转基因阳性纯合编辑。与CK相比,突变体（zmptac2）叶绿体发异常,叶绿素含量显著降低。RNA-seq的结果显示,CK与zmptac2之间共检测到1 367个基因差异表达,其中618个基因上调表达（zmptac2/CK）,749个基因下调表达（zmptac2/CK）。GO富集分析显示,下调基因显著富集到叶绿体和质体中。KEGG分析表明下调表达基因显著富集在苯丙氨酸代谢、酪氨酸代谢和异喹啉生物碱生物合成等途径。选取的15个差异基因表达模式均与测序数据相一致,表明测序结果是可靠的。与CK相比,zmptac2中依赖PEP（plastid-encoded RNA polymerase）转录的基因表达量显著降低,而依赖NEP（nuclear gene-encoded RNA polymerase）转录的基因表达量则显著上升。通过对玉米cDNA文库筛选和互作验证,鉴定出ZmpTAC3与ZmpTAC2存在互作。【结论】ZmpTAC2突变会导致叶绿体早期生物合成受阻,该基因参与叶绿体发育及叶绿素合成,且该种作用是由ZmpTAC2调控PEP相关基因表达而实现的。

关键词: 玉米, 叶绿素, ZmpTAC2, RNA-seq, 基因表达调控

Abstract:

【Objective】Chlorophyll is the most important photosynthetic pigment involved in plant photosynthesis. The development of chloroplasts and the synthesis of chlorophyll depend on the bi-directional signaling between the plastid and the nuclear genome. Plastid transcriptionally active chromosome proteins (pTACs) are essential for maintaining the transcriptional activity of PEP (plastid-encoded RNA polymerase) genes, whereas the function of pTACs in maize is still poorly understood. 【Method】The CRISPR/Cas9-ZmpTAC2 maize transgenic editing homozygous mutants were supplied. Transmission electron microscope were used to observe the ultrastructure of the chloroplast, with spectrophotometer to measure the chlorophyll content in maize leaves, respectively. Transcriptome sequencing of the negative material (CK) and CRISPR/Cas9-ZmpTAC2 transgenic homozygous (zmptac2) seedling leaves was performed. Bioinformatics toolbox was performed to identify the differently expressed genes between CK and zmptac2 leaves. Relative quantification of expression of selected differently expressed genes were verified using qRT-PCR. The interacting proteins of ZmpTAC2 were screened by yeast two-hybrid system. 【Result】 A total of 15 T₀transgenic seedlings were obtained, including 7 with green leaves and 8 with white ones. Among the seven green-leaf seedlings, 3 were transgenic negative together with 4 transgenic positive including 2 seedlings were edited. On the other hand, all 8 white-leaf seedlings were transgenic positive with homozygous editing. Compared with CK, zmptac2 chloroplast was abnormal with significantly reduced chlorophyll content. The results of RNA-seq showed that 1 367 genes were differentially expressed between CK and zmptac2, of which 618 genes were up-regulated (zmptac2/CK) and 749 genes down-regulated (zmptac2/CK). GO enrichment analysis revealed that the down-regulated genes were significantly enriched in chloroplasts and plastids. KEGG analysis indicated that down-regulated genes were abundant in the pathways of phenylalanine, tyrosine, and iso-quinoline alkaloid metabolism. Relative expression values of 15 selected differently expressed genes showed similar expression patterns and are consistent with sequencing data, which indicated that the sequencing results were reliable. The expression of PEP (plastid-encoded RNA polymerase) dependent genes in zmptac2 was significantly decreased, while the expression of NEP (nuclear gene-encoded RNA polymerase) genes increased. ZmpTAC3 was identified to be interacted with ZmpTAC2 by cDNA screening and verified by interaction assay. 【Conclusion】 This study first reported that mutations in the ZmpTAC2 gene cause early chloroplast biosynthesis to be hindered, indicating that this gene is involved in chloroplast development and chlorophyll synthesis, which is achieved by ZmpTAC2 regulated PEP-related gene expression.

Key words: maize (Zea mays L.), chlorophyll content, ZmpTAC2, RNA-seq, gene expression regulation

张稳,孟淑君,王琪月,万炯,马拴红,林源,丁冬,汤继华. 玉米pTAC2影响苗期叶片叶绿素合成的转录组分析[J]. 中国农业科学, 2020, 53(5): 874-889.

ZHANG Wen,MENG ShuJun,WANG QiYue,WAN Jiong,MA ShuanHong,LIN Yuan,DING Dong,TANG JiHua. Transcriptome Analysis of Maize pTAC2 Effects on Chlorophyll Synthesis in Seedling Leaves[J]. Scientia Agricultura Sinica, 2020, 53(5): 874-889.

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https://www.chinaagrisci.com/CN/Y2020/V53/I5/874

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酵母双杂筛库获得ZmpTAC2的互作蛋白质"

编号 No.	基因编号 Gene ID_v4	亚细胞定位 Subcellular localization	基因描述 Gene description
1	Zm00001d022242	细胞膜Cell membrane	胞间连丝胼胝质结合蛋白5 PLASMODESMATA CALLOSE-BINDING PROTEIN 5
2	Zm00001d010039	细胞壁Cell wall	含纤维素酶的蛋白质Cellulase containing protein
3	Zm00001d023305	叶绿体Chloroplast	Protein LURP-one-related 2
4	Zm00001d049153	叶绿体Chloroplast	催化/水解酶Catalytic/ hydrolase
5	Zm00001d041550	叶绿体Chloroplast	热休克蛋白3 Heat shock 70 kDa protein 3
6	Zm00001d035270	叶绿体Chloroplast	SAM结构域蛋白Sterile alpha motif (SAM) domain-containing protein
7	Zm00001d028221	叶绿体Chloroplast	ATP合酶4 ATP synthase4
8	Zm00001d046073	叶绿体Chloroplast	Protein trichome birefringence-like 26
9	Zm00001d032373	叶绿体Chloroplast	叶绿体RNA剪接体1 Chloroplast RNA splicing1
10	Zm00001d007417	叶绿体Chloroplast	ATP依赖性DNA解旋酶ATP-dependent DNA helicase chloroplastic
11	Zm00001d024248	叶绿体Chloroplast	PPR蛋白Pentatricopeptide repeat*-59040
12	Zm00001d016660	细胞质Cytoplasm	肽基脯氨酰异构酶1 Peptidyl-prolyl isomerase1
13	Zm00001d033623	细胞质Cytoplasm	脂氧合酶 Lipoxygenase
14	Zm00001d018441	细胞质,细胞核 Cytoplasm. Nucleus	核苷三磷酸水解酶超家族蛋白 P-loop containing nucleoside triphosphate hydrolases superfamily protein
15	Zm00001d040681	细胞质,细胞核 Cytoplasm. Nucleus	alpha/beta水解超家族蛋白 Alpha/beta-Hydrolases superfamily protein
16	Zm00001d038857	线粒体Mitochondrion	伴侣蛋白CPN60 Chaperonin CPN60-like 2 mitochondrial
17	Zm00001d035201	线粒体Mitochondrion	60S核糖体蛋白P060S acidic ribosomal protein P0
18	Zm00001d053434	细胞核Nucleus	RING锌指结构域超家族蛋白 Putative RING zinc finger domain superfamily protein
19	Zm00001d032422	细胞核Nucleus	休眠相关蛋白1 Dormancy-associated protein 1
20	Zm00001d044246	细胞核Nucleus	组蛋白H2A Histone H2A
21	Zm00001d044429	细胞核Nucleus	硫氧还蛋白超家族蛋白 Thioredoxin superfamily protein
22	Zm00001d021954	细胞核Nucleus	MO25蛋白 Putative MO25-like protein
23	Zm00001d032646	细胞核Nucleus	RHC1A
24	Zm00001d016876	细胞核Nucleus	Trihelix转录因子ASIL2 Trihelix transcription factor ASIL2
25	Zm00001d043914	细胞核Nucleus	Homeobox-DDT结构域蛋白RLT1 Homeobox-DDT domain protein RLT1
26	Zm00001d014858	细胞核Nucleus	DNA结合蛋白MNB1B DNA-binding protein MNB1B
27	Zm00001d036494	细胞核Nucleus	GATA转录因子25 GATA transcription factor 25
28	Zm00001d013159	细胞核Nucleus	α-微管蛋白3 Alpha-tubulin 3
29	Zm00001d001847	细胞核Nucleus	NUMOD3基序家族蛋白 NUMOD3 motif family protein expressed; protein
30	Zm00001d032790	细胞核Nucleus	质体转录活性3 Plastid transcriptionally active 3
31	Zm00001d052298	细胞核Nucleus	锌指（C3HC4型RING指）家族蛋白 Zinc finger (C3HC4-type RING finger) family protein
32	Zm00001d027652	液泡Vacuole	水通道蛋白TIP1-1 Aquaporin TIP1-1

表4

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编号 Serial number	潜在脱靶位置 Putative off-target locus	潜在脱靶序列 Sequence of the putative off-target site (5′-3′)	错配碱基数 No. of mismatching bases	测试株数 T₀ No. of plants tested
Off-target1	Chr.7:109756398-109756420	AgCTGGaGTAtAGCCTtATACGG	4	8
Off-target2	Chr.1 :145160658-145160680	AcCTGGGGTACAGCCaGATcAGG	3	8
Off-target3	Chr.6:44283532-44283554	cGaCCACGCAGCTATGTcTGAGG	3	8
Off-target4	Chr.4:230687431-230687453	cGaCCACGCAGCTATGTcTGAGG	3	8
Off-target5	Chr.7:8936191-8936213	TGGCCACGCgGCTAcGTgTGGGG	3	8
Off-target6	Chr.7:8936289-8936311	TGGCCACGCgGCTAcGTgTGGGG	3	8

样品 Sample	总碱基数 Total bases (Billion)	GC含量 GC percentage (%)	Q30 比例 Q30 percentage (%)	比对序列比例 Total mapping reads percentage (%)
CK_1	15	47.52	92.90	98.71
CK_2	16	47.50	91.94	98.75
zmptac2_1	12	47.52	90.29	98.87
zmptac2_2	16	47.92	92.17	98.56

基因编号 Gene ID	zmptac2	CK	差异倍数 log₂Fold change	P值 P-value	基因名称 Synonyms	描述 Description
Zm00001d039206	189.7162285	4.75059079	5.370830095	7.82E-06	Bzip118	bZIP转录因子超家族蛋白 Putative bZIP transcription factor superfamily protein
Zm00001d043825	80.63331881	0	8.789501589	0.00185833	CtpA1	羧基末端加工肽酶1 Carboxyl-terminal-processing peptidase 1 chloroplastic
Zm00001d027476	176.7085969	0	9.921454443	4.88E-06	DNA polymerases	依赖DNA的DNA聚合酶 DNA-directed DNA polymerases
Zm00001d031503	1000.059518	22.40077408	5.473911936	0.00680168	CPN60	伴侣蛋白亚基 Chaperonin 60 subunit alpha 2 chloroplastic
Zm00001d008221	0	351.1761292	-21.80949	1.54E-09	Cyc5	细胞周期蛋白5 Cyclin5
Zm00001d022599	363.2261699	3395.326772	-3.03756614	5.64E-14	ZmpTAC2	质体转录活性染色体2 Plastid transcriptionally active chromosome 2 homolog
Zm00001d033365	0	362.4571584	-23.97922	0.0000335	Cyclin A3 1	细胞周期蛋白A3-1 Putative cyclin-A3-1
Zm00001d027540	0	1170.714751	-25.15007	0.0000113	Gst12	谷胱甘肽S-转移酶12 Glutathione S-transferase12
Zm00001d048353	0	168.1203061	-22.56015	1.08E-08	Gst13	谷胱甘肽转移酶13 Glutathione transferase13
Zm00001d027760	0	1660.523115	-24.97806	1.32E-05	H2A	组蛋白H2A Histone H2A
Zm00001d051591	10.74539904	2639.275824	-7.938035	4.88E-05	his2b5	组蛋白2B5 Histone 2B5
Zm00001d042730	87.36474604	4052.072328	-5.535586	5.51E-05	H3	组蛋白H3 Histone H3
Zm00001d012276	103.9578986	6414.512969	-5.947576	0.000341	H3.2	组蛋白H3.2 Histone H3.2
Zm00001d039497	40.62122946	4971.062871	-6.93312404	8.97E-28	ClpC1	伴侣蛋白ClpC1（线粒体） Chaperone protein ClpC1 chloroplastic
Zm00001d042062	0	2272.297402	-9.395263783	0.00021753	ORG3	转录因子ORG3 Transcription factor ORG3