根系分隔方式下花魔芋转录组分析

doi:10.7668/hbnxb.20193441

摘要/Abstract

摘要：

了解与刺槐不同根系分隔方式下的花魔芋生物学过程及代谢通路,为间作花魔芋根系中相关基因的表达调控及响应机制提供分子基础。采用高通量测序平台Illumina NovaSeq 6000,对不同盆栽根系分隔(塑料膜分隔FS、尼龙网分隔MS和不分隔NS)方式下的花魔芋根系进行转录组测序分析。测序共获得59.82 Gb数据,组装后得到92 358条Transcript和40 122条Unigene序列,其中Nr数据库注释的信息最多(23 960条Unigene),占总Unigene的59.72%。将各样本获得的转录组数据进行两两比较,FS_vs_MS、FS_vs_NS和MS_vs_NS的差异表达基因数分别为1 776,733,896个,其中上调表达基因数量占差异表达基因比例分别为33.1%,22.8%和50.8%。GO功能富集分析显示,差异表达基因主要涉及细胞质膜、细胞膜、细胞外围、细胞壁、外部囊状结构、细胞膜内在成分及质膜内在成分等。KEGG富集分析表明,尼龙网分隔和未分隔间作花魔芋根系相对于塑料膜分隔单作花魔芋较显著的差异代谢通路在于核糖体、氧化磷酸化和植物激素信号转导。本研究通过花魔芋根系转录组分析,为研究间作花魔芋根系基因的表达调控机制提供基础数据资料。

关键词: 花魔芋, 间作, 转录组, 差异表达基因

Abstract:

The study aims to understand the biological process and metabolic pathway of konjac under different root separation modes from black locust,in order to provide molecular basis for the expression regulation and response mechanism of related genes in intercropping konjac roots.Illumina NovaSeq 6000 sequencing technology was used for transcriptome analysis of konjac roots separated by plastic membrane(FS),nylon mesh(MS),and non-separation(NS).A total of 59.82 Gb data were obtained,and 92 358 transcript sequences corresponding to 40 122 Unigenes were generated after de novo assembly and quantitative assessment.The most annotated Unigene in the NCBI-Nr database was 23 960,accounting for 59.72% of the total Unigenes.The transcriptome data were pair-wise compared,and the total quantities of differential expression genes of FS_vs_MS,FS_vs_NS and MS_vs_NS were 1 776,733 and 896,respectively.Of which,their corresponding up-regulated genes accounted for 33.1%,22.8% and 50.8%.GO enrichment analysis indicated that significantly differential expressed genes were mainly involved in plasma membrane,cell membrane,cell periphery,cell wall,encapsulating structure,intrinsic components of membrane,and intrinsic components of plasma membrane.KEGG enrichment analysis showed that the ribosome,oxidative phosphorylation and plant hormone signal transduction were the most significant metabolic pathways in the roots of konjac separated by nylon mesh and non-separation compared with plastic membrane.Transcriptome analysis of konjac roots provided the basic data for studying response mechanism of intercropping konjac.

Key words: Konjac, Intercropping, Transcriptome, DEGs

何斐, 李川, SHAH Faisal, 刘富强, 段园鹏, 王梦, 阮佳, 魏梦琳, 姜浩, 马星光, 王卓. 根系分隔方式下花魔芋转录组分析[J]. 华北农学报, 2023, 38(2): 65-74. doi: 10.7668/hbnxb.20193441.

HE Fei, LI Chuan, SHAH Faisal, LIU Fuqiang, DUAN Yuanpeng, WANG Meng, RUAN Jia, WEI Menglin, JIANG Hao, MA Xingguang, WANG Zhuo. Transcriptome Analysis of Amorphophallus konjac with Different Root Barriers[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 65-74. doi: 10.7668/hbnxb.20193441.

http://www.hbnxb.net/CN/Y2023/V38/I2/65

图/表 10

图1 两室根箱隔网系统

Fig.1 Two compartments system

表1 用于qRT-PCR验证的引物

Tab.1 Primers used for qRT-PCR validation

序号 No.	基因ID Gene ID	正向引物序列(5'-3') Forward primer sequence(5'-3')	反向引物序列(5'-3') Reverse primer sequence(5'-3')
1	TRINITY_DN5161_c0_g1	CTCTTCGTCTCGCTGGCAA	GGATGATCGGCATCGACCC
2	TRINITY_DN1055_c0_g1	AGTCGGACAACTGCTCCTTG	CCGAACACTTGTTGCCCTTG
3	TRINITY_DN41977_c0_g2	TGATGTGCACTAGCACCGAG	TAAGCCTGCTGGCATCATGT
4	TRINITY_DN1454_c0_g1	CCAGGCAGAAGAAGACCGTT	GCAGCGTACCACCAGTACAT
5	TRINITY_DN22369_c0_g1	CGGTGTCCGAAGACCATCAT	ATTTGGTTGAGGAGCTGGCA
6	TRINITY_DN1700_c0_g1	GTCCGCTTCTAGCACTCCTC	CACGAGATAGGCATCCGACC
7	EF1-a	AAGTTCCTGAAGAATGGCGAT	GTCCCTCACGGCAAACCTACC

表2 测序数据统计

Tab.2 Statistics of sequencing data

样品 Sample	原始序列数 Raw reads	原始碱基数 Raw bases	高质量序列 (占原始序列数比例/%) Clean reads (ratio)	高质量碱基 Clean bases	Q20/%	Q30/%
NS_1	46 227 980	6 934 197 000	40 747 808 (88.14)	6 112 171 200	96.55	92.35
NS_2	51 960 372	7 794 055 800	47 854 830 (92.09)	7 178 224 500	96.80	92.67
NS_3	56 569 902	8 485 485 300	52 236 882 (92.34)	7 835 532 300	96.54	92.08
MS_1	45 908 894	6 886 334 100	41 795 366 (91.03)	6 269 304 900	96.82	92.66
MS_2	46 103 366	6 915 504 900	41 337 718 (89.66)	6 200 657 700	96.79	92.64
MS_3	52 066 606	7 809 990 900	45 207 718 (86.82)	6 781 157 700	96.44	92.08
FS_1	50 236 112	7 535 416 800	44 801 948 (89.18)	6 720 292 200	96.50	92.04
FS_2	44 203 706	6 630 555 900	40 561 598 (91.76)	6 084 239 700	96.89	92.81
FS_3	49 690 406	7 453 560 900	44 279 090 (89.10)	6 641 863 500	96.49	92.22

图2 基于皮尔逊相关系数的样品间重复性分析对原始测序数据去除接头序列及低质量reads,共获得59.82 Gb Clean data,Trinity组装后共获得92 358条Transcript和40 122条Unigene序列,平均长度分别为1 047.36,912.36 bp,N50长度分别为1 478,1 441 bp,GC含量分别为48.49%和49.36%(表3)。

Fig.2 Repeatability analysis between samples based on Pearson correlation coefficient

表3 组装结果分析

Tab.3 Analysis of assembly

长度范围 Length range	转录本 Transcript	单一基因 Unigene
总数量Total number	92 358.00	40 122.00
总长度/bp Total length	96 731 890.00	36 605 858.00
N50长度/bp N50 length	1 478.00	1 441.00
平均长度/bp Mean length	1 047.36	912.36
GC/%	48.49	49.36

图3 处理间差异表达基因的火山图与统计分析

Fig.3 The volcano plot and statistical analysis of DEGs between treatments

图4 处理间差异表达基因的GO注释分析 1.生物过程;2.细胞进程;3.新陈代谢过程;4.有机物代谢过程;5.细胞代谢过程;6.初级代谢过程;7.氮化合物代谢过程;8.刺激响应;9.细胞组分;10.有机氮化合物代谢过程;11.细胞;12.细胞内;13.细胞器;14.细胞内细胞器;15.细胞质;16.膜结合细胞器;17.细胞内膜结合细胞器;18.细胞膜;19.分子功能;20.催化活性。

Fig.4 The GO annotations analysis of DEGs between treatments 1.Biological process;2.Cellular process;3.Metabolic process;4.Organic substance metabolic process;5.Cellular metabolic process;6.Primary metabolic process;7.Nitrogen compound metabolic process;8.Response to stimulus;9.Cellular component;10.Organonitrogen compound metabolic process;11.Cell;12.Intracellular;13.Organelle;14.Intracellular organelle;15.Cytoplasm;16.Membrane-bounded organelle;17.Intracellular membrane-bounded organelle;18.Membrane;19.Molecular function;20.Catalytic activity.

图5 处理间差异表达基因的GO富集分析

Fig.5 The GO enrichment analysis of DEGs between treatments

图6 处理间差异表达基因的KEGG富集分析

Fig.6 The KEGG enrichment analysis of DEGs between treatments

图7 6个差异表达基因qRT-PCR验证 1.阿拉伯半乳聚糖蛋白;2.磷脂酶A2;3.三角状五肽重复蛋白;4.水孔蛋白PIP2-8;5.DNA损伤修复蛋白;6.受体胞质丝氨酸/苏氨酸蛋白激酶RBK2。

Fig.7 qRT-PCR analysis of six selected DEGs 1.Arabinogalactan protein;2.Phospholipase A2;3.Pentatricopeptide repeat-containing protein;4.Aquaporin PIP2-8;5.DNA damage repair protein;6.Receptor-like cytosolic serine/threonine-protein kinase RBK2.

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