生物技术通报 ›› 2023, Vol. 39 ›› Issue (3): 254-266.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0773
扈丽丽1,2(), 林柏荣2,3, 王宏洪4, 陈建松2,3, 廖金铃4(), 卓侃2,3()
收稿日期:
2022-06-25
出版日期:
2023-03-26
发布日期:
2023-04-10
通讯作者:
廖金铃,男,博士,教授,研究方向:植物病原线虫学;E-mail: jlliao@scau.edu.cn;作者简介:
扈丽丽,女,博士,副研究员,研究方向:植物病原线虫学及林业有害生物综合防控;E-mail: hulili0113@163.com
基金资助:
HU Li-li1,2(), LIN Bo-rong2,3, WANG Hong-hong4, CHEN Jian-song2,3, LIAO Jin-ling4(), ZHUO Kan2,3()
Received:
2022-06-25
Published:
2023-03-26
Online:
2023-04-10
摘要:
最短尾短体线虫(Pratylenchus brachyurus)是一种重要的迁移性内寄生植物病原线虫。对其转录组进行测序和分析,是进一步研究其寄生特点和致病机理的重要基础。通过转录组测序和分析,得到72 516个unigenes,其中38 266个(52.76%)unigenes注释到7个蛋白质数据库中。Cazyme分析预测到 541 条具有植物/真菌细胞壁降解活性的蛋白质序列,分别属于 GH5、GH30、GH53、GH28、PL3、GH16、GH18和GH20家族。转录组中有56个蛋白序列与秀丽隐杆线虫(Caenorhabditis elegans)的RNAi通路蛋白具有同源性。鉴定到51个与已知植物寄生线虫效应蛋白具有同源性的蛋白序列,检测了9个潜在效应蛋白在抑制植物防卫反应中的作用,其中7个能够抑制由Gpa2/Rbp-1引起的细胞程序性死亡反应。研究结果表明最短尾短体线虫转录组与同属的短体线虫基因分布特点相似,其潜在效应蛋白具有抑制植物防卫反应的特点。
扈丽丽, 林柏荣, 王宏洪, 陈建松, 廖金铃, 卓侃. 最短尾短体线虫转录组及潜在效应蛋白分析[J]. 生物技术通报, 2023, 39(3): 254-266.
HU Li-li, LIN Bo-rong, WANG Hong-hong, CHEN Jian-song, LIAO Jin-ling, ZHUO Kan. Transcriptome and Candidate Effectors Analysis of Pratylenchus brachyurus[J]. Biotechnology Bulletin, 2023, 39(3): 254-266.
Primer name | Primer sequence(5'-3') | Application |
---|---|---|
45363F | ATGCTGTTCACCGCACTGCT | ORF amplification |
45363R | TTATGTTCTCTTCACCTCGC | ORF amplification |
39081F | ATGGTCTTCAAAAGCGCTTC | ORF amplification |
39081R | CTAATGCTGTTTCATCATTC | ORF amplification |
38325F | ATGCGTGCCAATTTGTTGTG | ORF amplification |
38325R | TCAATTGAGGCAATCACGCT | ORF amplification |
40687F | ATGCATCTAGTTCTATTACT | ORF amplification |
40687R | TCAGTCACGTTTGGCCAACT | ORF amplification |
41828F | ATGTTCCGCTGCTCTTCCTC | ORF amplification |
41828R | TCATTCCTCCTCTCGGTTCT | ORF amplification |
49596F | ATGGGCATTGGCTTGGCCAT | ORF amplification |
49596R | TCAGACCAACGGGTACGACG | ORF amplification |
50445F | ATGGCTACATCAATTTTTGT | ORF amplification |
50445R | TCAAAAAGCGATGAATGCGA | ORF amplification |
51705F | ATGCTTTCTTGCCGTCTCCTT | ORF amplification |
51705R | TCAAATGGGATGATTCAACA | ORF amplification |
41042F | ATGTTCTTTACTGTTGCTGG | ORF amplification |
41042R | TCAAATTCGAAAGCGTTCAA | ORF amplification |
828-1305F | TGACCATGGAAATTCCTGCTCGACAATTTGG | Vector construction |
828-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCTTCCTCCTCTCGGTTCTCTC | Vector construction |
363-1305F | TGACCATGGAACAACAGGAAAAATATCAGCC | Vector construction |
363-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCTGTTCTCTTCACCTCGCCCT | Vector construction |
081-1305F | TGACCATGGAA ATCGTCAGTGCGCTTCCCCG | Vector construction |
081-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCATGCTGTTTCATCATTCCTG | Vector construction |
325-1305F | TGACCATGGAA ATTGGCATTGGCCGAACGCA | Vector construction |
325-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCATTGAGGCAATCACGCTCCT | Vector construction |
687-1305F | TGACCATGGAA GCCAGCTGTGATTCGCCAAA | Vector construction |
687-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCGTCACGTTTGGCCAACTGTT | Vector construction |
596-1305F | TGACCATGGAA GTGCAATGGCGTGTTGAGCG | Vector construction |
596-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCGACCAACGGGTACGACGCCT | Vector construction |
445-1305F | TGACCATGGAA CAATCTCCCAATGCAGATAA | Vector construction |
445-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCAAAAGCGATGAATGCGACAG | Vector construction |
705-1305F | TGACCATGGAA GAACAATACGATTCAACGGA | Vector construction |
705-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCAATGGGATGATTCAACAGGA | Vector construction |
042-1305F | TGACCATGGAA CTGGGCTGTATGAGCACAAT | Vector construction |
042-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCAATTCGAAAGCGTTCAATCA | Vector construction |
表1 本研究所用引物列表
Table 1 Primers used in this study
Primer name | Primer sequence(5'-3') | Application |
---|---|---|
45363F | ATGCTGTTCACCGCACTGCT | ORF amplification |
45363R | TTATGTTCTCTTCACCTCGC | ORF amplification |
39081F | ATGGTCTTCAAAAGCGCTTC | ORF amplification |
39081R | CTAATGCTGTTTCATCATTC | ORF amplification |
38325F | ATGCGTGCCAATTTGTTGTG | ORF amplification |
38325R | TCAATTGAGGCAATCACGCT | ORF amplification |
40687F | ATGCATCTAGTTCTATTACT | ORF amplification |
40687R | TCAGTCACGTTTGGCCAACT | ORF amplification |
41828F | ATGTTCCGCTGCTCTTCCTC | ORF amplification |
41828R | TCATTCCTCCTCTCGGTTCT | ORF amplification |
49596F | ATGGGCATTGGCTTGGCCAT | ORF amplification |
49596R | TCAGACCAACGGGTACGACG | ORF amplification |
50445F | ATGGCTACATCAATTTTTGT | ORF amplification |
50445R | TCAAAAAGCGATGAATGCGA | ORF amplification |
51705F | ATGCTTTCTTGCCGTCTCCTT | ORF amplification |
51705R | TCAAATGGGATGATTCAACA | ORF amplification |
41042F | ATGTTCTTTACTGTTGCTGG | ORF amplification |
41042R | TCAAATTCGAAAGCGTTCAA | ORF amplification |
828-1305F | TGACCATGGAAATTCCTGCTCGACAATTTGG | Vector construction |
828-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCTTCCTCCTCTCGGTTCTCTC | Vector construction |
363-1305F | TGACCATGGAACAACAGGAAAAATATCAGCC | Vector construction |
363-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCTGTTCTCTTCACCTCGCCCT | Vector construction |
081-1305F | TGACCATGGAA ATCGTCAGTGCGCTTCCCCG | Vector construction |
081-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCATGCTGTTTCATCATTCCTG | Vector construction |
325-1305F | TGACCATGGAA ATTGGCATTGGCCGAACGCA | Vector construction |
325-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCATTGAGGCAATCACGCTCCT | Vector construction |
687-1305F | TGACCATGGAA GCCAGCTGTGATTCGCCAAA | Vector construction |
687-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCGTCACGTTTGGCCAACTGTT | Vector construction |
596-1305F | TGACCATGGAA GTGCAATGGCGTGTTGAGCG | Vector construction |
596-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCGACCAACGGGTACGACGCCT | Vector construction |
445-1305F | TGACCATGGAA CAATCTCCCAATGCAGATAA | Vector construction |
445-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCAAAAGCGATGAATGCGACAG | Vector construction |
705-1305F | TGACCATGGAA GAACAATACGATTCAACGGA | Vector construction |
705-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCAATGGGATGATTCAACAGGA | Vector construction |
042-1305F | TGACCATGGAA CTGGGCTGTATGAGCACAAT | Vector construction |
042-1305R | TCACACGTGTTACTTATCGTCGTCATCCTTGTAATCAATTCGAAAGCGTTCAATCA | Vector construction |
Data | Total reads/bp | Total base pairs/bp | Average length/bp | Length range/bp | N50/bp | N90/bp |
---|---|---|---|---|---|---|
Raw reads | 33 691 610 | 4 211 451 250 | - | - | - | - |
Clean reads>200 bp | 33 268 909 | 4 158 613 625 | - | - | - | - |
Transcripts | 101 595 | 80 122 468 | 789 | 200-15 754 | 1 284 | 1 131 |
Unigenes | 72 516 | 50 829 031 | 701 | 200-15 754 | 311 | 276 |
表2 最短尾短体线虫转录组数据概况
Table 2 Overview of transcriptome data of P. brachyurus transcriptome
Data | Total reads/bp | Total base pairs/bp | Average length/bp | Length range/bp | N50/bp | N90/bp |
---|---|---|---|---|---|---|
Raw reads | 33 691 610 | 4 211 451 250 | - | - | - | - |
Clean reads>200 bp | 33 268 909 | 4 158 613 625 | - | - | - | - |
Transcripts | 101 595 | 80 122 468 | 789 | 200-15 754 | 1 284 | 1 131 |
Unigenes | 72 516 | 50 829 031 | 701 | 200-15 754 | 311 | 276 |
Sequence file | NR | NT | Swiss-Prot | KEGG | PFAM | GO | KOG | ALL |
---|---|---|---|---|---|---|---|---|
Number | 31 374 | 3 314 | 24 164 | 13 521 | 29 265 | 29 729 | 19 183 | 72 516 |
Percentage/% | 43.26 | 4.57 | 33.32 | 18.64 | 40.35 | 40.99 | 26.45 | 100 |
表3 不同数据库中蛋白注释数量
Table 3 Statistics of proteins annotated in different database
Sequence file | NR | NT | Swiss-Prot | KEGG | PFAM | GO | KOG | ALL |
---|---|---|---|---|---|---|---|---|
Number | 31 374 | 3 314 | 24 164 | 13 521 | 29 265 | 29 729 | 19 183 | 72 516 |
Percentage/% | 43.26 | 4.57 | 33.32 | 18.64 | 40.35 | 40.99 | 26.45 | 100 |
CAZy enzyme classes | CAZy families | Number of proteins |
---|---|---|
Auxiliary activities(AA) | 8 | 64 |
Glycoside hydrolases(GH) | 56 | 1 285 |
Polysaccharide lyases(PLs) | 4 | 27 |
Carbohydrate esterases(CEs) | 9 | 280 |
Glycosyl transferases(GT) | 51 | 941 |
Carbohydrate-bind modules(CBMs) | 24 | 1 089 |
Total | 152 | 3 686 |
表4 最短尾短体线虫转录组中碳水化合物活性酶分析
Table 4 Carbohydrate-active enzyme analysis in P. brachyurus
CAZy enzyme classes | CAZy families | Number of proteins |
---|---|---|
Auxiliary activities(AA) | 8 | 64 |
Glycoside hydrolases(GH) | 56 | 1 285 |
Polysaccharide lyases(PLs) | 4 | 27 |
Carbohydrate esterases(CEs) | 9 | 280 |
Glycosyl transferases(GT) | 51 | 941 |
Carbohydrate-bind modules(CBMs) | 24 | 1 089 |
Total | 152 | 3 686 |
Species | Cellulose | Xylan | Arabinogalactan | Pectin | 1,3-Glucan | Chitin | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
GH5 | GH45 | GH30 | GH53 | GH28 | PL3 | GH16 | GH18 | GH19 | GH20 | |||||||
Pratylenchus brachyurus | 85 | 0 | 117 | 2 | 55 | 5 | 56 | 204 | 0 | 17 | ||||||
P. thornei | 2 | 1 | 2 | 0 | 0 | 2 | 0 | 11 | 2 | 3 | ||||||
P. coffeae | 15 | 0 | 1 | 1 | 2 | 4 | 1 | 0 | 0 | 0 | ||||||
P. zeae | 8 | 0 | 1 | 0 | 0 | 1 | 1 | 5 | 0 | 2 | ||||||
P. penetrans | 21 | 0 | 3 | 1 | 4 | 5 | 0 | 10 | 0 | 6 | ||||||
Aphelenchoides besseyi | - | + | - | - | - | - | + | - | - | + | ||||||
A. ritzemabosi | 4 | 7 | 6 | 0 | 0 | 0 | 15 | 68 | 11 | 4 | ||||||
Bursaphelenchus xylophilus | 0 | 11 | 0 | 0 | 0 | 15 | 6 | 9 | 2 | 7 | ||||||
Globodera pallida | 15 | 0 | 0 | 2 | 0 | 7 | - | - | - | - | ||||||
Heterodera avenae | 16 | 0 | 0 | 0 | 0 | 2 | 1 | 0 | 1 | 1 | ||||||
H. schachtii | 24 | 0 | 2 | 3 | 1 | 9 | 0 | 57 | 0 | 5 | ||||||
Meloidogyne incognita | 21 | 0 | 6 | 0 | 2 | 30 | 0 | 3 | 2 | 2 | ||||||
M. hapla | 6 | 0 | 1 | 0 | 2 | 22 | 0 | 4 | 0 | 1 | ||||||
Caenorhabditis elegans | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 38 | 6 | 5 |
表5 最短尾短体线与其他植物寄生线虫植物/真菌细胞壁降解酶比较分析
Table 5 Comparison of predicted plant/fungal cell wall degrading enzymes in P. brachyurus and other nematodes
Species | Cellulose | Xylan | Arabinogalactan | Pectin | 1,3-Glucan | Chitin | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
GH5 | GH45 | GH30 | GH53 | GH28 | PL3 | GH16 | GH18 | GH19 | GH20 | |||||||
Pratylenchus brachyurus | 85 | 0 | 117 | 2 | 55 | 5 | 56 | 204 | 0 | 17 | ||||||
P. thornei | 2 | 1 | 2 | 0 | 0 | 2 | 0 | 11 | 2 | 3 | ||||||
P. coffeae | 15 | 0 | 1 | 1 | 2 | 4 | 1 | 0 | 0 | 0 | ||||||
P. zeae | 8 | 0 | 1 | 0 | 0 | 1 | 1 | 5 | 0 | 2 | ||||||
P. penetrans | 21 | 0 | 3 | 1 | 4 | 5 | 0 | 10 | 0 | 6 | ||||||
Aphelenchoides besseyi | - | + | - | - | - | - | + | - | - | + | ||||||
A. ritzemabosi | 4 | 7 | 6 | 0 | 0 | 0 | 15 | 68 | 11 | 4 | ||||||
Bursaphelenchus xylophilus | 0 | 11 | 0 | 0 | 0 | 15 | 6 | 9 | 2 | 7 | ||||||
Globodera pallida | 15 | 0 | 0 | 2 | 0 | 7 | - | - | - | - | ||||||
Heterodera avenae | 16 | 0 | 0 | 0 | 0 | 2 | 1 | 0 | 1 | 1 | ||||||
H. schachtii | 24 | 0 | 2 | 3 | 1 | 9 | 0 | 57 | 0 | 5 | ||||||
Meloidogyne incognita | 21 | 0 | 6 | 0 | 2 | 30 | 0 | 3 | 2 | 2 | ||||||
M. hapla | 6 | 0 | 1 | 0 | 2 | 22 | 0 | 4 | 0 | 1 | ||||||
Caenorhabditis elegans | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 38 | 6 | 5 |
Caenorhab- ditis elegans | Meloidogyne incognita | Meloidog-yne hapla | Globodera pallida | Heterode-ra avenae | Bursaphelenc-hus xylophilus | Aphelencho-ides besseyi | Pratylenc- hus coffeae | Pratylench- us penetrans | Pratylenchus brachyurus | |
---|---|---|---|---|---|---|---|---|---|---|
Small RNA biosynthesis | 9 | 7 | 6 | 6 | 6 | 8 | 7 | 6 | 7 | 7 |
dsRNA uptake and spreading | 13 | 4 | 4 | 4 | 4 | 6 | 6 | 7 | 4 | 7 |
AGOs and RISC | 32 | 9 | 7 | 9 | 8 | 12 | 27 | 14 | 7 | 25 |
RNAi inhibitors | 9 | 2 | 3 | 2 | 3 | 2 | 5 | 1 | 2 | 6 |
Nuclear effectors | 15 | 6 | 7 | 5 | 8 | 9 | 12 | 6 | 7 | 9 |
Total | 78 | 28 | 27 | 26 | 29 | 37 | 57 | 34 | 27 | 54 |
表6 最短尾短体线虫RNAi通路相关蛋白与其他植物寄生线虫的比较分析
Table 6 Proteins involved in the RNAi pathway of P. brachyurus compared with other nematodes
Caenorhab- ditis elegans | Meloidogyne incognita | Meloidog-yne hapla | Globodera pallida | Heterode-ra avenae | Bursaphelenc-hus xylophilus | Aphelencho-ides besseyi | Pratylenc- hus coffeae | Pratylench- us penetrans | Pratylenchus brachyurus | |
---|---|---|---|---|---|---|---|---|---|---|
Small RNA biosynthesis | 9 | 7 | 6 | 6 | 6 | 8 | 7 | 6 | 7 | 7 |
dsRNA uptake and spreading | 13 | 4 | 4 | 4 | 4 | 6 | 6 | 7 | 4 | 7 |
AGOs and RISC | 32 | 9 | 7 | 9 | 8 | 12 | 27 | 14 | 7 | 25 |
RNAi inhibitors | 9 | 2 | 3 | 2 | 3 | 2 | 5 | 1 | 2 | 6 |
Nuclear effectors | 15 | 6 | 7 | 5 | 8 | 9 | 12 | 6 | 7 | 9 |
Total | 78 | 28 | 27 | 26 | 29 | 37 | 57 | 34 | 27 | 54 |
Protein/Gene name | Accession number | Species | Number of unigenes | Best E-value | Bset Identity/% | Bit score | Alignment length/bp |
---|---|---|---|---|---|---|---|
14-3-3/14-3-3b product | AAL40719 | Meloidogyne incognita | 15 | 1.34e-138 | 81.12 | 386 | 233 |
10A06 | ACU12489 | Heterodera schachtii | 0 | ||||
16D10 | Q06JG6 | Meloidogyne incognita | 0 | ||||
19C07 | AAO85458 | Heterodera schachtii | 0 | ||||
Annexin 4C10 | AAN32888 | Heterodera glycines | 6 | 2.15e-96 | 45.82 | 285 | 323 |
Calreticulin(Mi-crt-1) | AAL40720 | Meloidogyne incognita | 6 | 1.06e-170 | 82.77 | 481 | 354 |
Cysteine protease(Mi-cpl-1) | CAD89795 | Meloidogyne incognita | 54 | 0 | 80.25 | 561 | 324 |
Fatty acid retinoid binding(Gp-far-1) | CAA70477 | Globodera pallida | 9 | 2.73e-83 | 75.46 | 241 | 163 |
Glutathione-S-transferase(Mi-gsts-1) | ABN64198 | Meloidogyne incognita | 74 | 9.84e-92 | 60.70 | 263 | 201 |
Peroxiredoxin(Gr-TpX) | CAB48391 | Globodera rostochiensis | 9 | 9.76e-122 | 80.81 | 339 | 198 |
RAN-BP-like(Gr-A18) | CAC21848 | Globodera rostochiensis | 9 | 4.91e-24 | 37.21 | 88.2 | 129 |
RAN-BP-like(Gp-rbp-1) | AAV34698 | Globodera pallida | 14 | 1.07e-140 | 81.66 | 403 | 229 |
Glutathione peroxidase | CAD38523 | Globodera rostochiensis | 0 | ||||
Transthyretin-like protein(Rs-ttl-1, Rs-ttl-2, Rs-ttl-3, Rs-ttl-4) | CAM84510 | Radopholus similis | 58 | 2.27e-64 | 78.40 | 190 | 125 |
Ubiquitin extension protein(Hs-ubi-1) | AAP30081 | Heterodera schachtii | 26 | 2.96e-50 | 94.74 | 149 | 76 |
Venom allergen-like protein(Vap-1) | AAK60209 | Heterodera glycines | 4 | 2.41e-33 | 50.00 | 111 | 118 |
Venom allergen-like protein(Mi-mps-1) | AAD01511 | Meloidogyne incognita | 0 | ||||
7E12 | AAQ10021 | Meloidogyne incognita | 0 | ||||
Acid phosphatase | AAN08587 | Meloidogyne incognita | 36 | 0 | 56.89 | 523 | 450 |
Chitinase | AAN14978 | Heterodera glycines | 15 | 6.17e-69 | 40.41 | 219 | 344 |
Chorismate mutase 1 | ABB02655 | Meloidogyne arenaria | 1 | 8.78e-12 | 34.34 | 53.5 | 99 |
CLE peptide | AAO33474 | Heterodera glycines | 0 | ||||
ERp99 | AAG21337 | Heterodera glycines | 9 | 3.33e-54 | 75.81 | 177 | 124 |
Galectin | AAB61596 | Globodera rostochiensis | 27 | 1.16e-75 | 60.87 | 223 | 161 |
Map-1 | CAC27774 | Meloidogyne incognita | 0 | ||||
SPRYSEC(RBP-1) | CAM33004 | Globodera pallida | 2 | 3.09e-15 | 45.16 | 59.3 | 62 |
RING-H2 zinc finger protein | AAP30834 | Heterodera glycines | 0 | ||||
SKP1-like protein | AAP30763 | Heterodera glycines | 8 | 3.98e-30 | 35.36 | 105 | 181 |
SXP/RAL-2 | CAB75701 | Globodera rostochiensis | 4 | 4.87e-17 | 32.74 | 65.5 | 113 |
Ubiquitin extension protein | AAO33478 | Heterodera glycines | 1 | 2.03e-10 | 33.72 | 50.4 | 86 |
Venom allergen-like protein(VAP-1) | AEL16453 | Globodera rostochiensis | 8 | 1.24e-83 | 68.68 | 243 | 182 |
Effector protein GPP | ARC52277 | Meloidogyne graminicola | 1 | 3.99e-17 | 33.33 | 70.1 | 150 |
Secretory protein(MJ-NULG1a) | AFB73917 | Meloidogyne javanica | 0 | ||||
Cellulase(β-1,4-endo glucanase) | CAJ77137 | Meloidogyne javanica | 30 | 5.15e-129 | 72.13 | 380 | 302 |
表7 最短尾短体线虫与已知植物寄生线虫同源性分析
Table 7 Homology analysis between P. brachyurus and known plant-parasitic nematodes
Protein/Gene name | Accession number | Species | Number of unigenes | Best E-value | Bset Identity/% | Bit score | Alignment length/bp |
---|---|---|---|---|---|---|---|
14-3-3/14-3-3b product | AAL40719 | Meloidogyne incognita | 15 | 1.34e-138 | 81.12 | 386 | 233 |
10A06 | ACU12489 | Heterodera schachtii | 0 | ||||
16D10 | Q06JG6 | Meloidogyne incognita | 0 | ||||
19C07 | AAO85458 | Heterodera schachtii | 0 | ||||
Annexin 4C10 | AAN32888 | Heterodera glycines | 6 | 2.15e-96 | 45.82 | 285 | 323 |
Calreticulin(Mi-crt-1) | AAL40720 | Meloidogyne incognita | 6 | 1.06e-170 | 82.77 | 481 | 354 |
Cysteine protease(Mi-cpl-1) | CAD89795 | Meloidogyne incognita | 54 | 0 | 80.25 | 561 | 324 |
Fatty acid retinoid binding(Gp-far-1) | CAA70477 | Globodera pallida | 9 | 2.73e-83 | 75.46 | 241 | 163 |
Glutathione-S-transferase(Mi-gsts-1) | ABN64198 | Meloidogyne incognita | 74 | 9.84e-92 | 60.70 | 263 | 201 |
Peroxiredoxin(Gr-TpX) | CAB48391 | Globodera rostochiensis | 9 | 9.76e-122 | 80.81 | 339 | 198 |
RAN-BP-like(Gr-A18) | CAC21848 | Globodera rostochiensis | 9 | 4.91e-24 | 37.21 | 88.2 | 129 |
RAN-BP-like(Gp-rbp-1) | AAV34698 | Globodera pallida | 14 | 1.07e-140 | 81.66 | 403 | 229 |
Glutathione peroxidase | CAD38523 | Globodera rostochiensis | 0 | ||||
Transthyretin-like protein(Rs-ttl-1, Rs-ttl-2, Rs-ttl-3, Rs-ttl-4) | CAM84510 | Radopholus similis | 58 | 2.27e-64 | 78.40 | 190 | 125 |
Ubiquitin extension protein(Hs-ubi-1) | AAP30081 | Heterodera schachtii | 26 | 2.96e-50 | 94.74 | 149 | 76 |
Venom allergen-like protein(Vap-1) | AAK60209 | Heterodera glycines | 4 | 2.41e-33 | 50.00 | 111 | 118 |
Venom allergen-like protein(Mi-mps-1) | AAD01511 | Meloidogyne incognita | 0 | ||||
7E12 | AAQ10021 | Meloidogyne incognita | 0 | ||||
Acid phosphatase | AAN08587 | Meloidogyne incognita | 36 | 0 | 56.89 | 523 | 450 |
Chitinase | AAN14978 | Heterodera glycines | 15 | 6.17e-69 | 40.41 | 219 | 344 |
Chorismate mutase 1 | ABB02655 | Meloidogyne arenaria | 1 | 8.78e-12 | 34.34 | 53.5 | 99 |
CLE peptide | AAO33474 | Heterodera glycines | 0 | ||||
ERp99 | AAG21337 | Heterodera glycines | 9 | 3.33e-54 | 75.81 | 177 | 124 |
Galectin | AAB61596 | Globodera rostochiensis | 27 | 1.16e-75 | 60.87 | 223 | 161 |
Map-1 | CAC27774 | Meloidogyne incognita | 0 | ||||
SPRYSEC(RBP-1) | CAM33004 | Globodera pallida | 2 | 3.09e-15 | 45.16 | 59.3 | 62 |
RING-H2 zinc finger protein | AAP30834 | Heterodera glycines | 0 | ||||
SKP1-like protein | AAP30763 | Heterodera glycines | 8 | 3.98e-30 | 35.36 | 105 | 181 |
SXP/RAL-2 | CAB75701 | Globodera rostochiensis | 4 | 4.87e-17 | 32.74 | 65.5 | 113 |
Ubiquitin extension protein | AAO33478 | Heterodera glycines | 1 | 2.03e-10 | 33.72 | 50.4 | 86 |
Venom allergen-like protein(VAP-1) | AEL16453 | Globodera rostochiensis | 8 | 1.24e-83 | 68.68 | 243 | 182 |
Effector protein GPP | ARC52277 | Meloidogyne graminicola | 1 | 3.99e-17 | 33.33 | 70.1 | 150 |
Secretory protein(MJ-NULG1a) | AFB73917 | Meloidogyne javanica | 0 | ||||
Cellulase(β-1,4-endo glucanase) | CAJ77137 | Meloidogyne javanica | 30 | 5.15e-129 | 72.13 | 380 | 302 |
Protein number | Length /aa | Best matches in Nr | Species | E-value | Query cover/% | Identity /% | Accession Number |
---|---|---|---|---|---|---|---|
c40687_g1 | 184 | Translocon associated protein delta subunit | Pratylenchus goodeyi | 4e-80 | 93 | 67 | AHB64350 |
c56651_g1 | 136 | Beta-1,4-endoglucanase | Aphelenchus avenae | 4e-41 | 96 | 51 | BAI44493 |
c40783_g1 | 638 | Matrix metalloproteinase | Globodera rostochiensis | 0.0 | 87 | 51 | AAR11447 |
c49911_g2 | 481 | Beta-1,4-endoglucanase | Pratylenchus penetrans | 0.0 | 92 | 75 | BAB68522 |
c23527_g1 | 78 | Transthyretin-like protein 4 | Meloidogyne javanica | 3e-12 | 76 | 52 | AKV89653 |
c61136_g1 | 88 | Beta-1,4-endoglucanase precursor | Globodera rostochiensis | 1e-16 | 64 | 56 | AAC48325 |
c44079_g2 | 119 | Beta-1,4-endoglucanase | Pratylenchus goodeyi | 2e-34 | 57 | 81 | AJD14760 |
c40872_g1 | 241 | Lysozyme | Ditylenchus destructor | 2e-07 | 79 | 28 | ADW77529 |
c50445_g1 | 493 | Acid phosphatase | Heterodera avenae | 1e-125 | 90 | 43 | AFQ55439 |
c45880_g1 | 457 | Acid phosphatase | Heterodera avenae | 2e-94 | 87 | 41 | AFQ55439 |
c45363_g1 | 266 | Secreted glutathione peroxidase | Globodera rostochiensis | 4e-142 | 95 | 75 | AHW98769 |
c35087_g1 | 383 | GHF5 endo-1,4-beta-glucanase precursor | Radopholus similis | 7e-107 | 78 | 53 | ABV54446 |
c27617_g1 | 209 | C-type lectin | Rotylenchulus reniformis | 8e-13 | 75 | 31 | AFM37305 |
c50084_g1 | 268 | Secreted glutathione peroxidase | Heterodera cruciferae | 3e-114 | 76 | 72 | AJE31857 |
c41828_g1 | 318 | SXP/RAL-2 protein | Meloidogyne incognita | 1e-23 | 43 | 36 | AAR35032 |
c44657_g1 | 420 | Cathepsin L-like cysteine proteinase | Ditylenchus destructor | 0.0 | 93 | 67 | ACT35690 |
c41337_g1 | 476 | Serine carboxypeptidases | Radopholus similis | 0.0 | 94 | 58 | AIC75882 |
c35220_g1 | 242 | Glutathione peroxidases | Ditylenchus destructor | 5e-129 | 92 | 76 | AFJ15101 |
c44519_g1 | 467 | Acid phosphatase | Heterodera avenae | 2e-90 | 97 | 36 | AFQ55439 |
c39081_g1 | 240 | Putative amphid protein | Globodera rostochiensis | 2e-37 | 66 | 43 | CAB66341 |
c43667_g1 | 176 | Trans-thyretin-related family domain family member | Aphelenchoides besseyi | 2e-46 | 75 | 49 | AGA60310 |
c47241_g1 | 445 | Acid phosphatase | Heterodera avenae | 4e-95 | 84 | 43 | AFQ55439 |
c30155_g1 | 175 | Endoglucanase-2 precursor | Pratylenchus vulnus | 4e-38 | 94 | 45 | CDM79919 |
c61123_g1 | 100 | Polygalacturonase | Meloidogyne incognita | 2e-19 | 91 | 47 | AAM28240 |
c50692_g1 | 664 | Heat shock protein 70-C | Ditylenchus destructor | 0.0 | 99 | 90 | AFL69919 |
c43858_g1 | 423 | Acid phosphatase | Heterodera avenae | 2e-70 | 95 | 35 | AFQ55439 |
c26240_g1 | 156 | Putative esophageal gland cell secretory protein 21 | Meloidogyne incognita | 7e-28 | 99 | 37 | AAN08587 |
c30506_g1 | 142 | Transthyretin-like protein 2 precursor | Radopholus similis | 7e-43 | 79 | 58 | CAM845111 |
c50669_g2 | 288 | Serine proteinase | Meloidogyne incognita | 8e-92 | 95 | 50 | ABQ02009 |
c45343_g9 | 139 | Hypothetical esophageal gland cell secretory protein 11 | Heterodera glycines | 6e-46 | 87 | 65 | AAF76925 |
c45839_g1 | 357 | Biotin synthase | Heterodera glycines | 0.0 | 91 | 74 | ACZ34281 |
c65044_g1 | 165 | Expansin B | Globodera tabacum | 7e-16 | 32 | 73 | AEU04757 |
c38872_g2 | 372 | Expansin B | Meloidogyne javanica | 2e-09 | 30 | 35 | ADX36366 |
c49407_g1 | 213 | VAP1 protein | Globodera rostochiensis | 6e-86 | 96 | 62 | AEL16453 |
c16433_g1 | 257 | C-type lectin | Rotylenchulus reniformis | 2e-11 | 77 | 31 | AFM37307 |
c49384_g1 | 404 | Cathepsin L-like cysteine proteinase | Ditylenchus destructor | 7e-153 | 99 | 58 | ACT35690 |
c25396_g1 | 154 | Acid phosphatase | Heterodera avenae | 2e-42 | 94 | 51 | AFQ55439 |
c40020_g1 | 158 | Trans-thyretin-related family domain family member | Aphelenchoides besseyi | 2e-46 | 86 | 50 | AGA60310 |
c19843_g1 | 108 | Gland-specific protein g4e02 | Heterodera glycines | 3e-16 | 51 | 60 | AAO33473 |
c51705_g1 | 525 | Serine carboxypeptidases | Radopholus similis | 0.0 | 87 | 83 | AIC75882 |
c44522_g1 | 272 | Venom allergen-like protein vap-2 | Ditylenchus destructor | 1e-48 | 72 | 45 | ADC35399 |
c41042_g1 | 392 | Putative esophageal gland cell secretory protein 26 | Meloidogyne incognita | 6e-105 | 61 | 62 | AAN15806 |
c31126_g1 | 213 | Matrix metalloproteinase | Globodera rostochiensis | 6e-25 | 78 | 37 | AAR11447 |
c49596_g1 | 394 | Putative cathepsin L protease | Meloidogyne incognita | 0.0 | 97 | 70 | CAD89795 |
c29740_g1 | 238 | Dual oxidase | Meloidogyne incognita | 2e-122 | 82 | 91 | AAY84711 |
c38325_g1 | 151 | Transthyretin-like protein 1 precursor | Radopholus similis | 9e-54 | 82 | 73 | CAM84510 |
c42933_g1 | 363 | Arginine kinase | Heterodera glycines | 0.0 | 86 | 87 | AAO49799 |
c47148_g1 | 610 | Acetylcholinesterase 3 | Ditylenchus destructor | 0.0 | 95 | 70 | ABQ58115 |
c51959_g1 | 673 | Heat shock protein 70-C | Ditylenchus destructor | 0.0 | 94 | 90 | AFL69919 |
c45122_g1 | 84 | Transthyretin-like protein 4 | Meloidogyne javanica | 1e-19 | 84 | 58 | AKV89653 |
c40147_g1 | 415 | VAP1 protein | Globodera rostochiensis | 7e-56 | 41 | 52 | AEL16453 |
表8 最短尾短体线虫转录组中潜在效应蛋白的筛选
Table 8 Screeing of putative effectors in P. brachyurus transcriptome
Protein number | Length /aa | Best matches in Nr | Species | E-value | Query cover/% | Identity /% | Accession Number |
---|---|---|---|---|---|---|---|
c40687_g1 | 184 | Translocon associated protein delta subunit | Pratylenchus goodeyi | 4e-80 | 93 | 67 | AHB64350 |
c56651_g1 | 136 | Beta-1,4-endoglucanase | Aphelenchus avenae | 4e-41 | 96 | 51 | BAI44493 |
c40783_g1 | 638 | Matrix metalloproteinase | Globodera rostochiensis | 0.0 | 87 | 51 | AAR11447 |
c49911_g2 | 481 | Beta-1,4-endoglucanase | Pratylenchus penetrans | 0.0 | 92 | 75 | BAB68522 |
c23527_g1 | 78 | Transthyretin-like protein 4 | Meloidogyne javanica | 3e-12 | 76 | 52 | AKV89653 |
c61136_g1 | 88 | Beta-1,4-endoglucanase precursor | Globodera rostochiensis | 1e-16 | 64 | 56 | AAC48325 |
c44079_g2 | 119 | Beta-1,4-endoglucanase | Pratylenchus goodeyi | 2e-34 | 57 | 81 | AJD14760 |
c40872_g1 | 241 | Lysozyme | Ditylenchus destructor | 2e-07 | 79 | 28 | ADW77529 |
c50445_g1 | 493 | Acid phosphatase | Heterodera avenae | 1e-125 | 90 | 43 | AFQ55439 |
c45880_g1 | 457 | Acid phosphatase | Heterodera avenae | 2e-94 | 87 | 41 | AFQ55439 |
c45363_g1 | 266 | Secreted glutathione peroxidase | Globodera rostochiensis | 4e-142 | 95 | 75 | AHW98769 |
c35087_g1 | 383 | GHF5 endo-1,4-beta-glucanase precursor | Radopholus similis | 7e-107 | 78 | 53 | ABV54446 |
c27617_g1 | 209 | C-type lectin | Rotylenchulus reniformis | 8e-13 | 75 | 31 | AFM37305 |
c50084_g1 | 268 | Secreted glutathione peroxidase | Heterodera cruciferae | 3e-114 | 76 | 72 | AJE31857 |
c41828_g1 | 318 | SXP/RAL-2 protein | Meloidogyne incognita | 1e-23 | 43 | 36 | AAR35032 |
c44657_g1 | 420 | Cathepsin L-like cysteine proteinase | Ditylenchus destructor | 0.0 | 93 | 67 | ACT35690 |
c41337_g1 | 476 | Serine carboxypeptidases | Radopholus similis | 0.0 | 94 | 58 | AIC75882 |
c35220_g1 | 242 | Glutathione peroxidases | Ditylenchus destructor | 5e-129 | 92 | 76 | AFJ15101 |
c44519_g1 | 467 | Acid phosphatase | Heterodera avenae | 2e-90 | 97 | 36 | AFQ55439 |
c39081_g1 | 240 | Putative amphid protein | Globodera rostochiensis | 2e-37 | 66 | 43 | CAB66341 |
c43667_g1 | 176 | Trans-thyretin-related family domain family member | Aphelenchoides besseyi | 2e-46 | 75 | 49 | AGA60310 |
c47241_g1 | 445 | Acid phosphatase | Heterodera avenae | 4e-95 | 84 | 43 | AFQ55439 |
c30155_g1 | 175 | Endoglucanase-2 precursor | Pratylenchus vulnus | 4e-38 | 94 | 45 | CDM79919 |
c61123_g1 | 100 | Polygalacturonase | Meloidogyne incognita | 2e-19 | 91 | 47 | AAM28240 |
c50692_g1 | 664 | Heat shock protein 70-C | Ditylenchus destructor | 0.0 | 99 | 90 | AFL69919 |
c43858_g1 | 423 | Acid phosphatase | Heterodera avenae | 2e-70 | 95 | 35 | AFQ55439 |
c26240_g1 | 156 | Putative esophageal gland cell secretory protein 21 | Meloidogyne incognita | 7e-28 | 99 | 37 | AAN08587 |
c30506_g1 | 142 | Transthyretin-like protein 2 precursor | Radopholus similis | 7e-43 | 79 | 58 | CAM845111 |
c50669_g2 | 288 | Serine proteinase | Meloidogyne incognita | 8e-92 | 95 | 50 | ABQ02009 |
c45343_g9 | 139 | Hypothetical esophageal gland cell secretory protein 11 | Heterodera glycines | 6e-46 | 87 | 65 | AAF76925 |
c45839_g1 | 357 | Biotin synthase | Heterodera glycines | 0.0 | 91 | 74 | ACZ34281 |
c65044_g1 | 165 | Expansin B | Globodera tabacum | 7e-16 | 32 | 73 | AEU04757 |
c38872_g2 | 372 | Expansin B | Meloidogyne javanica | 2e-09 | 30 | 35 | ADX36366 |
c49407_g1 | 213 | VAP1 protein | Globodera rostochiensis | 6e-86 | 96 | 62 | AEL16453 |
c16433_g1 | 257 | C-type lectin | Rotylenchulus reniformis | 2e-11 | 77 | 31 | AFM37307 |
c49384_g1 | 404 | Cathepsin L-like cysteine proteinase | Ditylenchus destructor | 7e-153 | 99 | 58 | ACT35690 |
c25396_g1 | 154 | Acid phosphatase | Heterodera avenae | 2e-42 | 94 | 51 | AFQ55439 |
c40020_g1 | 158 | Trans-thyretin-related family domain family member | Aphelenchoides besseyi | 2e-46 | 86 | 50 | AGA60310 |
c19843_g1 | 108 | Gland-specific protein g4e02 | Heterodera glycines | 3e-16 | 51 | 60 | AAO33473 |
c51705_g1 | 525 | Serine carboxypeptidases | Radopholus similis | 0.0 | 87 | 83 | AIC75882 |
c44522_g1 | 272 | Venom allergen-like protein vap-2 | Ditylenchus destructor | 1e-48 | 72 | 45 | ADC35399 |
c41042_g1 | 392 | Putative esophageal gland cell secretory protein 26 | Meloidogyne incognita | 6e-105 | 61 | 62 | AAN15806 |
c31126_g1 | 213 | Matrix metalloproteinase | Globodera rostochiensis | 6e-25 | 78 | 37 | AAR11447 |
c49596_g1 | 394 | Putative cathepsin L protease | Meloidogyne incognita | 0.0 | 97 | 70 | CAD89795 |
c29740_g1 | 238 | Dual oxidase | Meloidogyne incognita | 2e-122 | 82 | 91 | AAY84711 |
c38325_g1 | 151 | Transthyretin-like protein 1 precursor | Radopholus similis | 9e-54 | 82 | 73 | CAM84510 |
c42933_g1 | 363 | Arginine kinase | Heterodera glycines | 0.0 | 86 | 87 | AAO49799 |
c47148_g1 | 610 | Acetylcholinesterase 3 | Ditylenchus destructor | 0.0 | 95 | 70 | ABQ58115 |
c51959_g1 | 673 | Heat shock protein 70-C | Ditylenchus destructor | 0.0 | 94 | 90 | AFL69919 |
c45122_g1 | 84 | Transthyretin-like protein 4 | Meloidogyne javanica | 1e-19 | 84 | 58 | AKV89653 |
c40147_g1 | 415 | VAP1 protein | Globodera rostochiensis | 7e-56 | 41 | 52 | AEL16453 |
Protein number | Length/aa | Best matches in Nr |
---|---|---|
c41828_g1 | 318 | SXP/RAL-2 protein |
c38325_g1 | 151 | Transthyretin-like protein 1 precursor |
c45363_g1 | 266 | Secreted glutathione peroxidase |
c51705_g1 | 525 | Serine carboxypeptidases |
c40687_g1 | 184 | Translocon associated protein delta subunit |
c50445_g1 | 493 | Acid phosphatase |
c41042_g1 | 392 | Putative esophageal gland cell secretory protein 26 |
c39081_g1 | 240 | Putative amphid protein |
c49596_g1 | 394 | Putative cathepsin L protease |
表9 用于在烟草叶片中开展抑制植物细胞程序性死亡试验的潜在效应蛋白
Table 9 Putative effectors selected for testing potential abilities in suppressing programmed cell death in Nicotiana benthamiana
Protein number | Length/aa | Best matches in Nr |
---|---|---|
c41828_g1 | 318 | SXP/RAL-2 protein |
c38325_g1 | 151 | Transthyretin-like protein 1 precursor |
c45363_g1 | 266 | Secreted glutathione peroxidase |
c51705_g1 | 525 | Serine carboxypeptidases |
c40687_g1 | 184 | Translocon associated protein delta subunit |
c50445_g1 | 493 | Acid phosphatase |
c41042_g1 | 392 | Putative esophageal gland cell secretory protein 26 |
c39081_g1 | 240 | Putative amphid protein |
c49596_g1 | 394 | Putative cathepsin L protease |
图3 潜在效应蛋白抑制Gpa2/RBP-1引起的细胞程序性死亡 A:潜在效应蛋白抑制Gpa2/RBP-1引起的细胞程序性死亡坏死斑.最后一次注射试验后5 d统计坏死斑并拍照,括号中是产生坏死斑数量/注射总数,开展3次独立生物学重复;B:以潜在效应蛋白c39081_g1 和 c41042_g1为例展示抑制或不抑制细胞程序性死亡的实验.在烟草叶片上先注射buffer,或转化有 c39081_g1、c41042_g1、CEP12 和 pCAMBIA1305:flag的农杆菌菌株,24 h后注射转化有RBP-1/Gpa2的农杆菌,用western blot验证蛋白表达量。1,5: buffer→24 h→Gpa2/RBP-1; 2,6: pCAMBIA1305:flag→24 h→Gpa2/RBP-1; 3,7: GrCEP12→24 h→Gpa2/RBP-1; 4: c39081_g1→24 h→Gpa2/RBP-1; 8: c41042_g1→24 h→Gpa2/RBP-1
Fig. 3 Suppression of Gpa2/RBP-1-induced cell programming death by putative effectors A: Necrotic spot of candidate effectors in the assay of suppressing cell death triggered by Gpa2/RBP-1. The necrotic spot was scored, and photographs were taken at 5 d after the last infiltration. The numbers in parentheses indicate the number of infiltrated sites showing cell-death symptoms divided by the total number of infiltrated sites. Two independent assays were performed. B: Suppression or no suppression of PCD was shown by examples of c39081_g1 and c41042_g1. N. benthamiana leaves were infiltrated with buffer or Agrobacterium tumefaciens cells carrying c39081_g1 or c41042_g1, CEP12 and pCAMBIA1305:flag, followed 24 h later with A. tumefaciens carrying RBP-1/Gpa2. Protein expression was confirmed by Western blot
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