生物技术通报 ›› 2020, Vol. 36 ›› Issue (12): 42-53.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0241
收稿日期:
2020-03-09
出版日期:
2020-12-26
发布日期:
2020-12-22
作者简介:
鲁琳,女,副教授,研究方向:园林植物;E-mail:基金资助:
LU Lin1(), YANG Shang-yu2, LIU Wei-dong1, LU Li-ming2()
Received:
2020-03-09
Published:
2020-12-26
Online:
2020-12-22
摘要:
为了鉴定参与花烟草盐胁迫响应的活性氧清除相关基因,用200 mmol/L NaCl处理花烟草幼苗,并在处理后12 h采集其幼苗样本,提取总RNA,采用高通量测序技术,进行转录组测序。基于差异表达基因GO及KEGG分析的基础上,对参与其中的活性氧清除基因进行挖掘,利用qRT-PCR的方法验证转录组测序结果。结果表明,盐处理后,花烟草基因表达量变化在2倍以上的基因有7 239个(P<0.01),其中,上调表达基因4 037个,下调表达基因3 162个,将其功能归类于生物过程、细胞组分及分子功能三大类159个GO条目,并显著富集在12条KEGG代谢通路中。同时,在盐胁迫下,花烟草有45个活性氧清除相关基因的表达发生了显著改变,其中,上调表达基因28个,下调表达基因17个。此结果说明,活性氧的清除是花烟草抵御盐胁迫的重要机制之一。
鲁琳, 杨尚谕, 刘维东, 鲁黎明. 基于转录组测序花烟草响应盐胁迫活性氧清除相关基因的挖掘[J]. 生物技术通报, 2020, 36(12): 42-53.
LU Lin, YANG Shang-yu, LIU Wei-dong, LU Li-ming. Mining of Genes Related to Reactive Oxygen Species Scavenging in Response to Salt Stress in Nicotiana alata Based on Transcriptome Sequencing[J]. Biotechnology Bulletin, 2020, 36(12): 42-53.
序号 | 引物名称 | 引物序列(5'-3') |
---|---|---|
1 | GST1-F | CCGGCAATAAGCATGAAGAT |
GST1-R | TAGTATCCCCTTGGGTGTGG | |
2 | CAT1-F | CCAATAAGCTACCACTACGAAACG |
CAT1-R | GGGAAAGAGGAGTGAACCCATT | |
3 | POD42-F | AAGAGCCATCACTATCTGGTACAAAG |
POD42-R | CTGCCTCTCCGTCTGAACCT | |
4 | CIPK11-F | GCGTTGACGGAGCAAGTACA |
CIPK11-R | ATGCTGGCGTTCCACAAAG | |
5 | ERF4-F | TCACTACGACTGGGCTTACCAA |
ERF4-R | GGAATTGCTCAATGTTTGTCCTAA | |
6 | GPX4-F | CCCTGATGTTCCACATGAGATCT |
GPX4-R | GGGTGGAAAGGAAACACAGTACA | |
7 | MYB5-F | GTGAAGGCCAATGGAGAAACTT |
MYB5-R | TCTTAGCCTACAACTTTTTCCACATC | |
8 | CIPK6-F | TCCGCCTTGGTTTTCATCAG |
CIPK6-R | TCGGATTCGGATCCAACATT | |
9 | Actin-F | CCAAGCAGCATGAAGATCAA |
Actin-R | CGTACTCGGCCTTTGAAATC |
表1 qPCR反应所用引物
序号 | 引物名称 | 引物序列(5'-3') |
---|---|---|
1 | GST1-F | CCGGCAATAAGCATGAAGAT |
GST1-R | TAGTATCCCCTTGGGTGTGG | |
2 | CAT1-F | CCAATAAGCTACCACTACGAAACG |
CAT1-R | GGGAAAGAGGAGTGAACCCATT | |
3 | POD42-F | AAGAGCCATCACTATCTGGTACAAAG |
POD42-R | CTGCCTCTCCGTCTGAACCT | |
4 | CIPK11-F | GCGTTGACGGAGCAAGTACA |
CIPK11-R | ATGCTGGCGTTCCACAAAG | |
5 | ERF4-F | TCACTACGACTGGGCTTACCAA |
ERF4-R | GGAATTGCTCAATGTTTGTCCTAA | |
6 | GPX4-F | CCCTGATGTTCCACATGAGATCT |
GPX4-R | GGGTGGAAAGGAAACACAGTACA | |
7 | MYB5-F | GTGAAGGCCAATGGAGAAACTT |
MYB5-R | TCTTAGCCTACAACTTTTTCCACATC | |
8 | CIPK6-F | TCCGCCTTGGTTTTCATCAG |
CIPK6-R | TCGGATTCGGATCCAACATT | |
9 | Actin-F | CCAAGCAGCATGAAGATCAA |
Actin-R | CGTACTCGGCCTTTGAAATC |
样品名称 | 原始数据 | 筛选后数据 | 总映射 | 多重映射 | 唯一映射 | Q30/% | GC含量/% |
---|---|---|---|---|---|---|---|
CK_1 | 49583710 | 47599094 | 44085400(92.62%) | 969397(2.04%) | 43116003(90.58%) | 90.32 | 42.98 |
CK_2 | 56621858 | 54483002 | 50100087(91.96%) | 1130193(2.07%) | 48969894(89.88%) | 90.99 | 43.03 |
CK_3 | 50230428 | 47972778 | 44659130(93.09%) | 965148(2.01%) | 43693982(91.08%) | 90.53 | 43.03 |
S12_1 | 64969188 | 62591696 | 57912308(92.52%) | 1294796(2.07%) | 56617512(90.46%) | 90.33 | 43.00 |
S12_2 | 52595884 | 50762784 | 47212736(93.01%) | 1022342(2.01%) | 46190394(90.99%) | 90.38 | 42.97 |
S12_3 | 53054528 | 51241836 | 47521067(92.74%) | 1099309(2.15%) | 46421758(90.59%) | 90.57 | 43.19 |
表2 各样品测序质量
样品名称 | 原始数据 | 筛选后数据 | 总映射 | 多重映射 | 唯一映射 | Q30/% | GC含量/% |
---|---|---|---|---|---|---|---|
CK_1 | 49583710 | 47599094 | 44085400(92.62%) | 969397(2.04%) | 43116003(90.58%) | 90.32 | 42.98 |
CK_2 | 56621858 | 54483002 | 50100087(91.96%) | 1130193(2.07%) | 48969894(89.88%) | 90.99 | 43.03 |
CK_3 | 50230428 | 47972778 | 44659130(93.09%) | 965148(2.01%) | 43693982(91.08%) | 90.53 | 43.03 |
S12_1 | 64969188 | 62591696 | 57912308(92.52%) | 1294796(2.07%) | 56617512(90.46%) | 90.33 | 43.00 |
S12_2 | 52595884 | 50762784 | 47212736(93.01%) | 1022342(2.01%) | 46190394(90.99%) | 90.38 | 42.97 |
S12_3 | 53054528 | 51241836 | 47521067(92.74%) | 1099309(2.15%) | 46421758(90.59%) | 90.57 | 43.19 |
GO分类号 | 功能描述 | 差异基因数量(DEGs) | 差异基因占比 /% |
---|---|---|---|
GO:0008150 | Biological_process | 4 595 | 76.97 |
GO:0008152 | Metabolic process | 3 660 | 61.31 |
GO:0044699 | Single-organism process | 2 808 | 47.04 |
GO:0044763 | Single-organism cellular process | 2 047 | 34.29 |
GO:0009058 | Biosynthetic process | 1 795 | 30.07 |
GO:0044710 | Single-organism metabolic process | 1 730 | 28.98 |
GO:0051234 | Establishment of localization | 1 121 | 18.78 |
GO:0006810 | Transport | 1 119 | 18.74 |
GO:1902578 | Single-organism localization | 845 | 14.15 |
GO:0044765 | Single-organism transport | 836 | 14.00 |
GO:0055114 | Oxidation-reduction process | 788 | 13.20 |
GO:0006796 | Phosphate-containing compound metabolic process | 693 | 11.61 |
GO:0006793 | Phosphorus metabolic process | 693 | 11.61 |
GO:0044711 | Single-organism biosynthetic process | 646 | 10.82 |
GO:0006464 | Cellular protein modification process | 563 | 9.43 |
GO:0036211 | Protein modification process | 563 | 9.43 |
GO:0005975 | Carbohydrate metabolic process | 549 | 9.20 |
GO:0044281 | Small molecule metabolic process | 535 | 8.96 |
GO:0006629 | Lipid metabolic process | 524 | 8.78 |
GO:0055085 | Transmembrane transport | 464 | 7.77 |
GO:0006811 | Ion transport | 460 | 7.71 |
GO:0016310 | Phosphorylation | 449 | 7.52 |
GO:1901135 | Carbohydrate derivative metabolic process | 428 | 7.17 |
GO:0044255 | Cellular lipid metabolic process | 366 | 6.13 |
GO:0006812 | Cation transport | 361 | 6.05 |
GO:0044723 | Single-organism carbohydrate metabolic process | 359 | 6.01 |
GO:0008610 | Lipid biosynthetic process | 320 | 5.36 |
GO:0019637 | Organophosphate metabolic process | 309 | 5.18 |
GO:1901137 | Carbohydrate derivative biosynthetic process | 267 | 4.47 |
GO:0044262 | Cellular carbohydrate metabolic process | 240 | 4.02 |
表3 上调差异表达基因的GO功能注释分析(生物过程)
GO分类号 | 功能描述 | 差异基因数量(DEGs) | 差异基因占比 /% |
---|---|---|---|
GO:0008150 | Biological_process | 4 595 | 76.97 |
GO:0008152 | Metabolic process | 3 660 | 61.31 |
GO:0044699 | Single-organism process | 2 808 | 47.04 |
GO:0044763 | Single-organism cellular process | 2 047 | 34.29 |
GO:0009058 | Biosynthetic process | 1 795 | 30.07 |
GO:0044710 | Single-organism metabolic process | 1 730 | 28.98 |
GO:0051234 | Establishment of localization | 1 121 | 18.78 |
GO:0006810 | Transport | 1 119 | 18.74 |
GO:1902578 | Single-organism localization | 845 | 14.15 |
GO:0044765 | Single-organism transport | 836 | 14.00 |
GO:0055114 | Oxidation-reduction process | 788 | 13.20 |
GO:0006796 | Phosphate-containing compound metabolic process | 693 | 11.61 |
GO:0006793 | Phosphorus metabolic process | 693 | 11.61 |
GO:0044711 | Single-organism biosynthetic process | 646 | 10.82 |
GO:0006464 | Cellular protein modification process | 563 | 9.43 |
GO:0036211 | Protein modification process | 563 | 9.43 |
GO:0005975 | Carbohydrate metabolic process | 549 | 9.20 |
GO:0044281 | Small molecule metabolic process | 535 | 8.96 |
GO:0006629 | Lipid metabolic process | 524 | 8.78 |
GO:0055085 | Transmembrane transport | 464 | 7.77 |
GO:0006811 | Ion transport | 460 | 7.71 |
GO:0016310 | Phosphorylation | 449 | 7.52 |
GO:1901135 | Carbohydrate derivative metabolic process | 428 | 7.17 |
GO:0044255 | Cellular lipid metabolic process | 366 | 6.13 |
GO:0006812 | Cation transport | 361 | 6.05 |
GO:0044723 | Single-organism carbohydrate metabolic process | 359 | 6.01 |
GO:0008610 | Lipid biosynthetic process | 320 | 5.36 |
GO:0019637 | Organophosphate metabolic process | 309 | 5.18 |
GO:1901137 | Carbohydrate derivative biosynthetic process | 267 | 4.47 |
GO:0044262 | Cellular carbohydrate metabolic process | 240 | 4.02 |
GO分类号 | 功能描述 | 差异基因数量(DEGs) | 差异基因占比/% |
---|---|---|---|
GO:0003824 | Catalytic activity | 3 314 | 55.51 |
GO:0016491 | Oxidoreductase activity | 852 | 14.27 |
GO:0005215 | Transporter activity | 658 | 11.02 |
GO:0022857 | Transmembrane transporter activity | 555 | 9.30 |
GO:0016301 | Kinase activity | 539 | 9.03 |
GO:0016773 | Phosphotransferase activity,alcohol group as acceptor | 516 | 8.64 |
GO:0022891 | Substrate-specific transmembrane transporter Activity | 437 | 7.32 |
GO:0004672 | Protein kinase activity | 413 | 6.92 |
GO:0048037 | Cofactor binding | 287 | 4.81 |
GO:0016705 | Oxidoreductase activity,acting on paired donors,with incorporation or reduction of molecular oxygen | 238 | 3.99 |
GO:0050662 | Coenzyme binding | 209 | 3.50 |
GO:0016614 | Oxidoreductase activity,acting on CH-OH group of donors | 168 | 2.81 |
GO:0020037 | Heme binding | 166 | 2.78 |
GO:0016616 | Oxidoreductase activity,acting on the CH-OH group of donors,NAD or NADP as acceptor | 163 | 2.73 |
GO:0005506 | Iron ion binding | 161 | 2.70 |
GO:0022804 | Active transmembrane transporter activity | 152 | 2.55 |
GO:0005509 | Calcium ion binding | 129 | 2.16 |
GO:0015291 | Secondary active transmembrane transporter activity | 84 | 1.41 |
GO:0016706 | Oxidoreductase activity,acting on paired donors,with incorporation or reduction of molecular oxygen,2-oxoglutarate as one donor,and incorporation of one atom each of oxygen into both donors | 70 | 1.17 |
GO:0004497 | Monooxygenase activity | 48 | 0.80 |
GO:0003857 | 3-hydroxyacyl-CoA dehydrogenase activity | 39 | 0.65 |
GO:0004616 | Phosphogluconate dehydrogenase(decarboxylating)activity | 36 | 0.60 |
GO:0016709 | Oxidoreductase activity,acting on paired donors,with incorporation or reduction of molecular oxygen,NAD(P)H as one donor,and incorporation of one atom of oxygen | 29 | 0.49 |
GO:0008677 | 2-dehydropantoate 2-reductase activity | 27 | 0.45 |
GO:0004312 | fatty acid synthase activity | 21 | 0.35 |
GO:0004315 | 3-oxoacyl-[acyl-carrier-protein]synthase activity | 20 | 0.34 |
GO:0016832 | Aldehyde-lyase activity | 19 | 0.32 |
GO:0004332 | Fructose-bisphosphate aldolase activity | 16 | 0.27 |
GO:0016872 | Intramolecular lyase activity | 13 | 0.22 |
GO:0004506 | Squalene monooxygenase activity | 13 | 0.22 |
GO:0003913 | DNA photolyase activity | 9 | 0.15 |
GO:0004512 | Inositol-3-phosphate synthase activity | 6 | 0.10 |
表4 上调差异表达基因的GO功能注释分析(分子功能)
GO分类号 | 功能描述 | 差异基因数量(DEGs) | 差异基因占比/% |
---|---|---|---|
GO:0003824 | Catalytic activity | 3 314 | 55.51 |
GO:0016491 | Oxidoreductase activity | 852 | 14.27 |
GO:0005215 | Transporter activity | 658 | 11.02 |
GO:0022857 | Transmembrane transporter activity | 555 | 9.30 |
GO:0016301 | Kinase activity | 539 | 9.03 |
GO:0016773 | Phosphotransferase activity,alcohol group as acceptor | 516 | 8.64 |
GO:0022891 | Substrate-specific transmembrane transporter Activity | 437 | 7.32 |
GO:0004672 | Protein kinase activity | 413 | 6.92 |
GO:0048037 | Cofactor binding | 287 | 4.81 |
GO:0016705 | Oxidoreductase activity,acting on paired donors,with incorporation or reduction of molecular oxygen | 238 | 3.99 |
GO:0050662 | Coenzyme binding | 209 | 3.50 |
GO:0016614 | Oxidoreductase activity,acting on CH-OH group of donors | 168 | 2.81 |
GO:0020037 | Heme binding | 166 | 2.78 |
GO:0016616 | Oxidoreductase activity,acting on the CH-OH group of donors,NAD or NADP as acceptor | 163 | 2.73 |
GO:0005506 | Iron ion binding | 161 | 2.70 |
GO:0022804 | Active transmembrane transporter activity | 152 | 2.55 |
GO:0005509 | Calcium ion binding | 129 | 2.16 |
GO:0015291 | Secondary active transmembrane transporter activity | 84 | 1.41 |
GO:0016706 | Oxidoreductase activity,acting on paired donors,with incorporation or reduction of molecular oxygen,2-oxoglutarate as one donor,and incorporation of one atom each of oxygen into both donors | 70 | 1.17 |
GO:0004497 | Monooxygenase activity | 48 | 0.80 |
GO:0003857 | 3-hydroxyacyl-CoA dehydrogenase activity | 39 | 0.65 |
GO:0004616 | Phosphogluconate dehydrogenase(decarboxylating)activity | 36 | 0.60 |
GO:0016709 | Oxidoreductase activity,acting on paired donors,with incorporation or reduction of molecular oxygen,NAD(P)H as one donor,and incorporation of one atom of oxygen | 29 | 0.49 |
GO:0008677 | 2-dehydropantoate 2-reductase activity | 27 | 0.45 |
GO:0004312 | fatty acid synthase activity | 21 | 0.35 |
GO:0004315 | 3-oxoacyl-[acyl-carrier-protein]synthase activity | 20 | 0.34 |
GO:0016832 | Aldehyde-lyase activity | 19 | 0.32 |
GO:0004332 | Fructose-bisphosphate aldolase activity | 16 | 0.27 |
GO:0016872 | Intramolecular lyase activity | 13 | 0.22 |
GO:0004506 | Squalene monooxygenase activity | 13 | 0.22 |
GO:0003913 | DNA photolyase activity | 9 | 0.15 |
GO:0004512 | Inositol-3-phosphate synthase activity | 6 | 0.10 |
GO分类号 | 功能描述 | 类型 | 差异基因数量(DEGs) | 差异基因占比/% |
---|---|---|---|---|
GO:0000041 | Transition metal ion transport | 生物过程 | 71 | 1.39 |
GO:0072511 | Divalent inorganic cation transport | 75 | 1.46 | |
GO:0070838 | Divalent metal ion transport | 59 | 1.15 | |
GO:0015684 | Ferrous iron transport | 41 | 0.80 | |
GO:0006826 | Iron ion transport | 44 | 0.86 | |
GO:0046915 | Transition metal ion transmembrane transporter activity | 分子功能 | 68 | 1.33 |
GO:0072509 | Divalent inorganic cation Transmembrane transporter activity | 75 | 1.46 | |
GO:0015093 | Ferrous iron transmembrane transporter activity | 41 | 0.80 | |
GO:0004748 | Ribonucleoside-diphosphate reductase activity,thioredoxin disulfide as acceptor | 14 | 0.27 | |
GO:0016728 | Oxidoreductase activity,acting on CH or CH2 groups,disulfide as acceptor | 14 | 0.27 | |
GO:0061731 | Ribonucleoside-diphosphate reductase activity | 14 | 0.27 | |
GO:0005381 | Iron ion transmembrane transporter activity | 44 | 0.86 |
表5 下调差异表达基因的GO功能注释分析
GO分类号 | 功能描述 | 类型 | 差异基因数量(DEGs) | 差异基因占比/% |
---|---|---|---|---|
GO:0000041 | Transition metal ion transport | 生物过程 | 71 | 1.39 |
GO:0072511 | Divalent inorganic cation transport | 75 | 1.46 | |
GO:0070838 | Divalent metal ion transport | 59 | 1.15 | |
GO:0015684 | Ferrous iron transport | 41 | 0.80 | |
GO:0006826 | Iron ion transport | 44 | 0.86 | |
GO:0046915 | Transition metal ion transmembrane transporter activity | 分子功能 | 68 | 1.33 |
GO:0072509 | Divalent inorganic cation Transmembrane transporter activity | 75 | 1.46 | |
GO:0015093 | Ferrous iron transmembrane transporter activity | 41 | 0.80 | |
GO:0004748 | Ribonucleoside-diphosphate reductase activity,thioredoxin disulfide as acceptor | 14 | 0.27 | |
GO:0016728 | Oxidoreductase activity,acting on CH or CH2 groups,disulfide as acceptor | 14 | 0.27 | |
GO:0061731 | Ribonucleoside-diphosphate reductase activity | 14 | 0.27 | |
GO:0005381 | Iron ion transmembrane transporter activity | 44 | 0.86 |
图3 花烟草响应高盐胁迫的抗氧化基因 GR:glutathione reductase;GST:glutathione S-transferases;APX:ascorbate peroxidase;GPX:glutathione peroxidase;CAT:Catalases;POD:peroxidase;SOD:Superoxide dismutase
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