镉胁迫下梭鱼草叶片转录组测序及苯丙烷代谢途径相关基因挖掘

doi:10.13560/j.cnki.biotech.bull.1985.2021-1151

摘要/Abstract

摘要：

为揭示苯丙烷代谢途径参与梭鱼草防御重金属胁迫的作用机制,分析镉（Cd）胁迫下梭鱼草叶片转录组序列,将其与NR、NT、PFAM、KOG、Swiss Prot、KEGG和GO公共数据库进行比对注释,并对差异表达基因进行趋势化分析,同时从中挖掘与苯丙烷代谢途径相关的差异表达基因。结果表明：（1）共获得221 392个unigenes,其中170 175个unigenes被注释到数据库,6 506个unigenes被注释到31条次生代谢通路;（2）将梭鱼草叶片unigenes与Swiss-Prot和Nr数据库进行比对,共得到168 355条CDS序列,Estscan软件预测得到84 673条序列;（3）对检测到的20 025个差异表达基因进行趋势分析,发现有3个显著的基因表达模式,包括2个下调表达模式（Cluster 0：2 631个基因,Cluster 1：3 153个基因）和1个上调表达模式（Cluster 5：3 733个基因）;（4）进一步挖掘转录组数据发现,在6_0 h和48_0 h组中,梭鱼草叶片中共有26个差异表达的基因被鉴定出来,分别编码3个苯丙烷代谢公共途径关键酶,5个木质素生物合成关键酶和7个黄酮类化合物生物合成关键酶。梭鱼草通过调控愈创木基木质素的生物合成和上调肉桂酸-4-羟化酶（C4H）、花青素合成酶（ANS）、花青素-3-O-葡糖基转移酶（3GT）、咖啡酰辅酶A-O甲基转移酶（CCoAOMT）、黄酮醇3-甲基转移酶（F3OMT）基因表达参与叶片Cd积累与解毒。通过转录组测序初步揭示了苯丙烷代谢途径参与梭鱼草叶片防御Cd胁迫的相关基因,为今后深入研究苯丙烷代谢途径在梭鱼草抵抗重金属胁迫中的功能及相关机制提供了理论基础。

关键词: 镉, 梭鱼草, 叶片转录组, 苯丙烷途径, 基因挖掘

Abstract:

To provide theoretical base for the mechanism of phenylpropane metabolic pathway involved in Pontederia cordata against heavy metal exposure,and to analyze the transcriptome sequences in the leaves of P. cordata exposed to cadmium（Cd）,the obtained Unigenes were compared and annotated with seven databases including NR,NT,PFAM,KOG,Swiss Prot,and KEGG as well as GO,and the tendency of differentially expressed genes（DEGs）were analyzed,and DEGs involved in phenypropanoid metabolic pathway were mined. Results showed as follows. 1）A total of 221 392 unigenes were obtained,of which 170 175 unigenes were successfully annotated in the public database,and 6 506 unigenes were annotated for 31 secondary metabolic pathways. 2）A total of 168 355 were obtained after comparing the Unigenes from the leaves of P. cordata with dadabase Swiss Prot and NR. And 84 673 CDS sequences were obtained by Esacan software. 3）Trend analysis demonstrated that 20 025 DEGs were clustered into three significant profiles including two down-regulated profiles（cluster 0 covering 2 631 DEGs and cluster 1 covering 3 153 DEGs）and one up-regulated profiles（cluster 5 covering 3 733 DEGs）. 4）With Cd²⁺ the exposure,there were 26 DEGs identified in P. cordata leaves in 6 h and 48 h by further mining the transcriptome data,which encoded 3 crucial enzymes in the common pathway of phenylpropane metabolism,5 crucial enzymes related to lignin biosynthesis,and 7 crucial enzymes responsible for flavonoid biosynthesis. P. cordata is involved in the accumulation and detoxification of Cd in the leaves by regulating the biosynthesis of guaiacyl lignin and up-regulating the gene expression of cinnamate-4-hydroxylase（C4H）,anthocyanin synthase（ANS）,anthocyanin-3-O -glucosyltransferase（3GT）,caffeonyl coenzyme A-O methyltransferase（CCoAOMT）,and flavonol 3-methyltransferase（F3OMT）. The genes involved in phenylpropane metabolic pathway in the leaves of P. cordata against Cd stress is preliminarily revealed by transcriptome sequencing,which provides a theoretical basis for further research on the function of phenylpropane metabolic pathway in the resistance of the plant to heavy metal stress and related mechanisms.

Key words: cadmium, Pontederia cordata, transcriptome in leaves, phenypropanoid pathway, gene mining

辛建攀, 李燕, 赵楚, 田如男. 镉胁迫下梭鱼草叶片转录组测序及苯丙烷代谢途径相关基因挖掘[J]. 生物技术通报, 2022, 38(6): 198-210.

XIN Jian-pan, LI Yan, ZHAO Chu, TIAN Ru-nan. Transcriptome Sequencing in the Leaves of Pontederia cordata with Cadmium Exposure and Gene Mining in Phenypropanoid Pathways[J]. Biotechnology Bulletin, 2022, 38(6): 198-210.

图/表 12

参考文献 43

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	Min length/bp	Max length/bp	Mean length/bp	N₅₀	N₉₀
Transcripts	201	25 638	1 542	2 554	746
Unigenes	201	25 638	1 753	2 609	883

	Min length/bp	Max length/bp	Mean length/bp	N₅₀	N₉₀
Transcripts	201	25 638	1 542	2 554	746
Unigenes	201	25 638	1 753	2 609	883

数据库 Annotated database	Unigenes数量 Number of unigenes	占比 Percentage/%
Annotated in NR	158 229	71.47
Annotated in NT	107 290	48.46
Annotated in KO	67 629	30.54
Annotated in SwissProt	121 487	54.87
Annotated in PFAM	98 391	44.44
Annotated in GO	98 391	44.44
Annotated in KOG	49 597	22.40
Annotated in all databases	22 729	10.26
Annotated in at least one database	170 175	76.86
Total unigenes	221 392	100

数据库 Annotated database	Unigenes数量 Number of unigenes	占比 Percentage/%
Annotated in NR	158 229	71.47
Annotated in NT	107 290	48.46
Annotated in KO	67 629	30.54
Annotated in SwissProt	121 487	54.87
Annotated in PFAM	98 391	44.44
Annotated in GO	98 391	44.44
Annotated in KOG	49 597	22.40
Annotated in all databases	22 729	10.26
Annotated in at least one database	170 175	76.86
Total unigenes	221 392	100

序号 No.	通路 Pathway	通路_ID Pathway_ID	Unigenes数量 Numbers of unigenes
1	苯丙素生物合成Phenylpropanoid biosynthesis	ko00940	696
2	缬氨酸、亮氨酸和异亮氨酸降解Valine,leucine and isoleucine degradation	ko00280	621
3	萜类化合物骨架生物合成Terpenoid backbone biosynthesis	ko00900	597
4	类胡萝卜素生物合成Carotenoid biosynthesis	ko00906	488
5	酪氨酸代谢Tyrosine metabolism	ko00350	426
6	泛醌及其他萜类醌生物合成Ubiquinone and other terpenoid-quinone biosynthesis	ko00130	370
7	苯丙氨酸、酪氨酸和色氨酸生物合成Phenylalanine,tyrosine and tryptophan biosynthesis	ko00400	346
8	烟酸和烟酰胺代谢Nicotinate and nicotinamide metabolism	ko00760	326
9	苯丙烷代谢Phenylalanine metabolism	ko00360	291
10	色氨酸代谢Tryptophan metabolism	ko00380	269
11	类固醇生物合成Steroid biosynthesis	ko00100	235
12	类黄酮生物合成Flavonoid biosynthesis	ko00941	222
13	异喹啉生物碱生物合成Isoquinoline alkaloid biosynthesis	ko00950	216
14	莨菪烷、哌啶和吡啶生物碱生物合成Tropane,piperidine and pyridine alkaloid biosynthesis	ko00960	197
15	玉米素生物合成Zeatin biosynthesis	ko00908	197
16	二苯乙烯、二芳基庚烷和姜酚生物合成Stilbenoid,diarylheptanoid and gingerol biosynthesis	ko00945	155
17	亮氨酸代谢Linoleic acid metabolism	ko00591	143
18	缬氨酸、亮氨酸和异亮氨酸生物合成Valine,leucine and isoleucine biosynthesis	ko00290	139
19	柠檬烯和蒎烯降解Limonene and pinene degradation	ko00903	128
20	油菜素内酯生物合成Brassinosteroid biosynthesis	ko00905	97
21	二萜生物合成Diterpenoid biosynthesis	ko00904	83
22	黄酮和黄酮醇生物合成Flavone and flavonol biosynthesis	ko00944	55
23	芥子油苷生物合成Glucosinolate biosynthesis	ko00966	58
24	单萜的生物合成Monoterpenoid biosynthesis	ko00902	48
25	倍半萜和三萜生物合成Sesquiterpenoid and triterpenoid biosynthesis	ko00909	44
26	咖啡因代谢Caffeine metabolism	ko00232	28
27	花青素生物合成Anthocyanin biosynthesis	ko00942	14
29	甜菜红碱生物合成Betalain biosynthesis	ko00965	10
30	异黄酮生物合成Isoflavonoid biosynthesis	ko00943	6
31	吲哚生物碱生物合成Indole alkaloid biosynthesis	ko00901	1