Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (6): 1213-1232.doi: 10.16420/j.issn.0513-353x.2021-0331
• Research Papers • Previous Articles Next Articles
ZHANG Kai, MA Mingying, WANG Ping, LI Yi, JIN Yan, SHENG Ling, DENG Ziniu, MA Xianfeng()
Received:
2021-12-14
Revised:
2022-02-25
Online:
2022-06-25
Published:
2022-07-04
Contact:
MA Xianfeng
E-mail:ma8006@hunau.edu.cn
CLC Number:
ZHANG Kai, MA Mingying, WANG Ping, LI Yi, JIN Yan, SHENG Ling, DENG Ziniu, MA Xianfeng. Identification of HSP20 Family Genes in Citrus and Their Expression in Pathogen Infection Responses Citrus Canker[J]. Acta Horticulturae Sinica, 2022, 49(6): 1213-1232.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2021-0331
目的基因引物 Primer | 上游引物序列(5′-3′) Forward primer sequence | 下游引物序列(5′-3′) Reverse primer sequence |
---|---|---|
EF-1α(qRT-PCR) | GTAACCAAGTCTGCTGCCAAG | GACCCAAACACCCAACACATT |
CsHSP17.9(qRT-PCR) | TGGTACGAAAGCAAACCCCT | TCAGACTTACAATTGCAAACACACA |
CmHSP17.9(qRT-PCR) | TGGGCCATAGAAGAAGCAGC | AGCATCCGTAACGACACCAA |
CsHSP23.3(qRT-PCR) | CTTCCGGGTGCTAGAGCAAA | CGTTTCCTCTCTCCGCTCAC |
CsHSP18.5(qRT-PCR) | TTTTCCATCTGCGGTGTCCT | CCTTAGCCCCGGAAGATCAG |
CsHSP18.0(qRT-PCR) | AAGCATTTCCGAAGTTGTTGAG | TTGGGATGTTTCCCTAGCCG |
CmHSP18.0(qRT-PCR) | TGGGCCATAGAAGAAGCAGC | AGCATCCGTAACGACACCAA |
CsHSP17.9(ORF) | TAGGTACCATGTCACTCATTCCAAGCATCT | TAGGTACCGCCAGAGATCTCAATAGCCTTA |
CmHSP17.9(ORF) | GGGAGCTCATGTCACTCATTCCAAGCATCT | AAGAGCTCAGAGCTGGCATCTGTTCCAGG |
CsHSP23.3(ORF) | TAGGTACCATGAGGCAGCAACAACAAACA | TAGGTACCAAGCTCTTGCCTAGCCTCGCTC |
CmHSP23.3(ORF) | TAGGTACCATGAGACAGCAACAACGAACAC | AAGGTACCAAGCTCTTGCCTAGCCTCGCTC |
CsHSP18.5(ORF) | TAGGTACCATGTCGCTGATTCCAAGCTTCT | TAGGTACCGCCAGAGATTTGAATAGCCCTG |
Table 1 Primer sequences used in this study
目的基因引物 Primer | 上游引物序列(5′-3′) Forward primer sequence | 下游引物序列(5′-3′) Reverse primer sequence |
---|---|---|
EF-1α(qRT-PCR) | GTAACCAAGTCTGCTGCCAAG | GACCCAAACACCCAACACATT |
CsHSP17.9(qRT-PCR) | TGGTACGAAAGCAAACCCCT | TCAGACTTACAATTGCAAACACACA |
CmHSP17.9(qRT-PCR) | TGGGCCATAGAAGAAGCAGC | AGCATCCGTAACGACACCAA |
CsHSP23.3(qRT-PCR) | CTTCCGGGTGCTAGAGCAAA | CGTTTCCTCTCTCCGCTCAC |
CsHSP18.5(qRT-PCR) | TTTTCCATCTGCGGTGTCCT | CCTTAGCCCCGGAAGATCAG |
CsHSP18.0(qRT-PCR) | AAGCATTTCCGAAGTTGTTGAG | TTGGGATGTTTCCCTAGCCG |
CmHSP18.0(qRT-PCR) | TGGGCCATAGAAGAAGCAGC | AGCATCCGTAACGACACCAA |
CsHSP17.9(ORF) | TAGGTACCATGTCACTCATTCCAAGCATCT | TAGGTACCGCCAGAGATCTCAATAGCCTTA |
CmHSP17.9(ORF) | GGGAGCTCATGTCACTCATTCCAAGCATCT | AAGAGCTCAGAGCTGGCATCTGTTCCAGG |
CsHSP23.3(ORF) | TAGGTACCATGAGGCAGCAACAACAAACA | TAGGTACCAAGCTCTTGCCTAGCCTCGCTC |
CmHSP23.3(ORF) | TAGGTACCATGAGACAGCAACAACGAACAC | AAGGTACCAAGCTCTTGCCTAGCCTCGCTC |
CsHSP18.5(ORF) | TAGGTACCATGTCGCTGATTCCAAGCTTCT | TAGGTACCGCCAGAGATTTGAATAGCCCTG |
基因名称 Gene name | CAP登录号 CAP ID | 染色体 Chr. | 基因位置 Genomic position | 开放阅读框 ORF | 外显子数 Exon number | 氨基酸数 Amino acid number | 分子量/ kD MW | 等电点 pI |
---|---|---|---|---|---|---|---|---|
CsHSP20-01 | Cs1g12560 | 1 | 15649712 ~ 15650511 (-) | 800 | 2 | 235 | 26.81 | 9.08 |
CsHSP20-02 | Cs1g16780 | 1 | 20054644 ~ 20057349 (-) | 539 | 3 | 135 | 15.17 | 9.01 |
CsHSP20-03 | Cs2g07920 | 2 | 4751770 ~ 4753509 (-) | 490 | 3 | 136 | 15.44 | 4.94 |
CsHSP20-04 | Cs2g24040 | 2 | 22865474 ~ 22866152 (+) | 417 | 1 | 138 | 15.65 | 5.79 |
CsHSP20-05 | Cs2g24360 | 2 | 23415680 ~ 23417091 (-) | 865 | 2 | 229 | 25.80 | 6.77 |
CsHSP20-06 | Cs2g28910 | 2 | 28489066 ~ 28490856 (+) | 1 630 | 2 | 308 | 34.20 | 9.26 |
CsHSP20-07 | Cs3g07660 | 3 | 10958719 ~ 10959189 (-) | 471 | 1 | 156 | 18.08 | 5.70 |
CsHSP20-08 | Cs3g13530 | 3 | 17951675 ~ 17954257 (+) | 2 280 | 3 | 208 | 22.15 | 4.81 |
CsHSP20-09 | Cs3g21390 | 3 | 24314830 ~ 24316680 (-) | 1 232 | 2 | 373 | 41.71 | 4.80 |
CsHSP20-10 | Cs4g04260 | 4 | 2440105 ~ 2443442 (-) | 453 | 1 | 150 | 16.84 | 4.53 |
CsHSP20-11 | Cs4g05780 | 4 | 3542671 ~ 3543153 (-) | 483 | 1 | 160 | 17.87 | 5.57 |
CsHSP20-12 | Cs4g05800 | 4 | 3545945 ~ 3546616 (-) | 489 | 1 | 162 | 18.39 | 6.45 |
CsHSP20-13 | Cs4g05880 | 4 | 3592334 ~ 3593451 (-) | 477 | 1 | 158 | 17.80 | 5.37 |
CsHSP20-14 | Cs4g12260 | 4 | 9326064 ~ 9327838 (+) | 773 | 2 | 198 | 22.48 | 5.48 |
CsHSP20-15 | Cs5g04230 | 5 | 2361326 ~ 2362866 (+) | 819 | 2 | 180 | 20.34 | 4.73 |
CsHSP20-16 | Cs5g18500 | 5 | 18849146 ~ 18850530 (+) | 612 | 1 | 203 | 23.26 | 5.77 |
CsHSP20-17 | Cs5g26930 | 5 | 29657216 ~ 29657893 (+) | 630 | 2 | 182 | 20.96 | 10.07 |
CsHSP20-18 | Cs5g26940 | 5 | 29661034 ~ 29661937 (+) | 715 | 2 | 207 | 23.08 | 9.10 |
CsHSP20-19 | Cs6g07310 | 6 | 9164497 ~ 9165164 (-) | 471 | 1 | 156 | 17.78 | 7.93 |
CsHSP20-20 | Cs6g07320 | 6 | 9166603 ~ 9167820 (+) | 438 | 1 | 145 | 16.34 | 7.92 |
CsHSP20-21 | Cs6g17370 | 6 | 17879116 ~ 17880307 (-) | 1 192 | 2 | 148 | 16.55 | 6.92 |
CsHSP20-22 | Cs7g10040 | 7 | 6437870 ~ 6438966 (-) | 779 | 2 | 224 | 25.41 | 8.67 |
CsHSP20-23 | Cs7g23870 | 7 | 22776659 ~ 22778441 (-) | 1 597 | 2 | 339 | 37.75 | 5.30 |
CsHSP20-24 | Cs7g23890 | 7 | 22813654 ~ 22814811 (-) | 1 062 | 2 | 309 | 34.41 | 8.20 |
CsHSP20-25 | Cs7g32260 | 7 | 31935895 ~ 31936951 (-) | 471 | 1 | 156 | 17.64 | 5.55 |
CsHSP20-26 | Cs8g13310 | 8 | 16029454 ~ 16030281 (+) | 828 | 1 | 275 | 30.11 | 5.68 |
CsHSP20-27 | Cs8g13370 | 8 | 16132947 ~ 16133375 (+) | 429 | 1 | 142 | 15.91 | 5.84 |
CsHSP20-28 | Cs8g13450 | 8 | 16211348 ~ 16212457 (+) | 1 110 | 1 | 369 | 40.91 | 5.97 |
CsHSP20-29 | Cs8g13500 | 8 | 16261568 ~ 16262230 (+) | 663 | 1 | 220 | 23.89 | 8.02 |
CsHSP20-30 | Cs8g13560 | 8 | 16334995 ~ 16335974 (+) | 980 | 1 | 224 | 24.62 | 5.75 |
CsHSP20-31 | Cs8g18020 | 8 | 20763153 ~ 20764015 (+) | 477 | 1 | 158 | 17.98 | 5.84 |
CsHSP20-32 | Cs8g18360 | 8 | 20993803 ~ 20994946 (-) | 477 | 1 | 158 | 17.89 | 6.77 |
CsHSP20-33 | Cs8g19490 | 8 | 21656820 ~ 21657642 (-) | 486 | 1 | 161 | 18.28 | 6.17 |
CsHSP20-34 | Cs8g19510 | 8 | 21667991 ~ 21668854 (-) | 486 | 1 | 161 | 18.22 | 6.76 |
CsHSP20-35 | Cs8g19520 | 8 | 21670200 ~ 21671498 (+) | 489 | 1 | 162 | 18.47 | 5.79 |
CsHSP20-36 | Cs8g19530 | 8 | 21674289 ~ 21675349 (+) | 492 | 1 | 163 | 18.46 | 6.76 |
CsHSP20-37 | Cs8g19540 | 8 | 21677280 ~ 21677985 (-) | 453 | 1 | 150 | 17.15 | 6.18 |
CsHSP20-38 | Cs9g14690 | 9 | 14093289 ~ 14096486 (-) | 1 036 | 2 | 207 | 23.37 | 5.43 |
CsHSP20-39 | orange1.1t01849 | Un | 29239328 ~ 29242931 (+) | 939 | 1 | 312 | 34.78 | 8.56 |
CsHSP20-40 | orange1.1t03235 | Un | 49911150 ~ 49912086 (+) | 489 | 1 | 162 | 17.98 | 7.13 |
CsHSP20-41 | orange1.1t03877 | Un | 59580055 ~ 59580573 (-) | 519 | 1 | 172 | 19.78 | 7.93 |
CsHSP20-42 | orange1.1t03881 | Un | 59667559 ~ 59668167 (-) | 609 | 1 | 202 | 23.10 | 6.61 |
Table 2 Features of HSP20 family genes in Citrus sinensis
基因名称 Gene name | CAP登录号 CAP ID | 染色体 Chr. | 基因位置 Genomic position | 开放阅读框 ORF | 外显子数 Exon number | 氨基酸数 Amino acid number | 分子量/ kD MW | 等电点 pI |
---|---|---|---|---|---|---|---|---|
CsHSP20-01 | Cs1g12560 | 1 | 15649712 ~ 15650511 (-) | 800 | 2 | 235 | 26.81 | 9.08 |
CsHSP20-02 | Cs1g16780 | 1 | 20054644 ~ 20057349 (-) | 539 | 3 | 135 | 15.17 | 9.01 |
CsHSP20-03 | Cs2g07920 | 2 | 4751770 ~ 4753509 (-) | 490 | 3 | 136 | 15.44 | 4.94 |
CsHSP20-04 | Cs2g24040 | 2 | 22865474 ~ 22866152 (+) | 417 | 1 | 138 | 15.65 | 5.79 |
CsHSP20-05 | Cs2g24360 | 2 | 23415680 ~ 23417091 (-) | 865 | 2 | 229 | 25.80 | 6.77 |
CsHSP20-06 | Cs2g28910 | 2 | 28489066 ~ 28490856 (+) | 1 630 | 2 | 308 | 34.20 | 9.26 |
CsHSP20-07 | Cs3g07660 | 3 | 10958719 ~ 10959189 (-) | 471 | 1 | 156 | 18.08 | 5.70 |
CsHSP20-08 | Cs3g13530 | 3 | 17951675 ~ 17954257 (+) | 2 280 | 3 | 208 | 22.15 | 4.81 |
CsHSP20-09 | Cs3g21390 | 3 | 24314830 ~ 24316680 (-) | 1 232 | 2 | 373 | 41.71 | 4.80 |
CsHSP20-10 | Cs4g04260 | 4 | 2440105 ~ 2443442 (-) | 453 | 1 | 150 | 16.84 | 4.53 |
CsHSP20-11 | Cs4g05780 | 4 | 3542671 ~ 3543153 (-) | 483 | 1 | 160 | 17.87 | 5.57 |
CsHSP20-12 | Cs4g05800 | 4 | 3545945 ~ 3546616 (-) | 489 | 1 | 162 | 18.39 | 6.45 |
CsHSP20-13 | Cs4g05880 | 4 | 3592334 ~ 3593451 (-) | 477 | 1 | 158 | 17.80 | 5.37 |
CsHSP20-14 | Cs4g12260 | 4 | 9326064 ~ 9327838 (+) | 773 | 2 | 198 | 22.48 | 5.48 |
CsHSP20-15 | Cs5g04230 | 5 | 2361326 ~ 2362866 (+) | 819 | 2 | 180 | 20.34 | 4.73 |
CsHSP20-16 | Cs5g18500 | 5 | 18849146 ~ 18850530 (+) | 612 | 1 | 203 | 23.26 | 5.77 |
CsHSP20-17 | Cs5g26930 | 5 | 29657216 ~ 29657893 (+) | 630 | 2 | 182 | 20.96 | 10.07 |
CsHSP20-18 | Cs5g26940 | 5 | 29661034 ~ 29661937 (+) | 715 | 2 | 207 | 23.08 | 9.10 |
CsHSP20-19 | Cs6g07310 | 6 | 9164497 ~ 9165164 (-) | 471 | 1 | 156 | 17.78 | 7.93 |
CsHSP20-20 | Cs6g07320 | 6 | 9166603 ~ 9167820 (+) | 438 | 1 | 145 | 16.34 | 7.92 |
CsHSP20-21 | Cs6g17370 | 6 | 17879116 ~ 17880307 (-) | 1 192 | 2 | 148 | 16.55 | 6.92 |
CsHSP20-22 | Cs7g10040 | 7 | 6437870 ~ 6438966 (-) | 779 | 2 | 224 | 25.41 | 8.67 |
CsHSP20-23 | Cs7g23870 | 7 | 22776659 ~ 22778441 (-) | 1 597 | 2 | 339 | 37.75 | 5.30 |
CsHSP20-24 | Cs7g23890 | 7 | 22813654 ~ 22814811 (-) | 1 062 | 2 | 309 | 34.41 | 8.20 |
CsHSP20-25 | Cs7g32260 | 7 | 31935895 ~ 31936951 (-) | 471 | 1 | 156 | 17.64 | 5.55 |
CsHSP20-26 | Cs8g13310 | 8 | 16029454 ~ 16030281 (+) | 828 | 1 | 275 | 30.11 | 5.68 |
CsHSP20-27 | Cs8g13370 | 8 | 16132947 ~ 16133375 (+) | 429 | 1 | 142 | 15.91 | 5.84 |
CsHSP20-28 | Cs8g13450 | 8 | 16211348 ~ 16212457 (+) | 1 110 | 1 | 369 | 40.91 | 5.97 |
CsHSP20-29 | Cs8g13500 | 8 | 16261568 ~ 16262230 (+) | 663 | 1 | 220 | 23.89 | 8.02 |
CsHSP20-30 | Cs8g13560 | 8 | 16334995 ~ 16335974 (+) | 980 | 1 | 224 | 24.62 | 5.75 |
CsHSP20-31 | Cs8g18020 | 8 | 20763153 ~ 20764015 (+) | 477 | 1 | 158 | 17.98 | 5.84 |
CsHSP20-32 | Cs8g18360 | 8 | 20993803 ~ 20994946 (-) | 477 | 1 | 158 | 17.89 | 6.77 |
CsHSP20-33 | Cs8g19490 | 8 | 21656820 ~ 21657642 (-) | 486 | 1 | 161 | 18.28 | 6.17 |
CsHSP20-34 | Cs8g19510 | 8 | 21667991 ~ 21668854 (-) | 486 | 1 | 161 | 18.22 | 6.76 |
CsHSP20-35 | Cs8g19520 | 8 | 21670200 ~ 21671498 (+) | 489 | 1 | 162 | 18.47 | 5.79 |
CsHSP20-36 | Cs8g19530 | 8 | 21674289 ~ 21675349 (+) | 492 | 1 | 163 | 18.46 | 6.76 |
CsHSP20-37 | Cs8g19540 | 8 | 21677280 ~ 21677985 (-) | 453 | 1 | 150 | 17.15 | 6.18 |
CsHSP20-38 | Cs9g14690 | 9 | 14093289 ~ 14096486 (-) | 1 036 | 2 | 207 | 23.37 | 5.43 |
CsHSP20-39 | orange1.1t01849 | Un | 29239328 ~ 29242931 (+) | 939 | 1 | 312 | 34.78 | 8.56 |
CsHSP20-40 | orange1.1t03235 | Un | 49911150 ~ 49912086 (+) | 489 | 1 | 162 | 17.98 | 7.13 |
CsHSP20-41 | orange1.1t03877 | Un | 59580055 ~ 59580573 (-) | 519 | 1 | 172 | 19.78 | 7.93 |
CsHSP20-42 | orange1.1t03881 | Un | 59667559 ~ 59668167 (-) | 609 | 1 | 202 | 23.10 | 6.61 |
Fig. 2 The phylogenetic relationships of HSP20 genes in different species Cs:C. sinensis;Cm:C. medica;At:Arabidopsis thaliana;Os:Oryza sativa;PGSC:Solanum tuberosum;VIT:Vitis vinifera. The numbers on the branch are bootstrap value(%),the larger the value,the higher the confidence of the evolutionary branch.
保守基序名称 Motif name | 氨基酸序列 Amino acid sequence | 起始位点 Site | 序列长度/aa Width |
---|---|---|---|
Motif 1 | WKETPEAHVFKADLPGLRKEEVKVE | 32 | 25 |
Motif 2 | KMDQIKASMENGVLTVTVPK | 33 | 20 |
Motif 3 | SSGMFSRRFRLPENA | 31 | 15 |
Motif 4 | VEDDRVLQISGQRKIEKEDKN | 27 | 21 |
Motif 5 | MSLIPSFFGNRRSSVFDPFSLDVWDPFRDF | 8 | 30 |
Motif 6 | DTWHRVER | 16 | 8 |
Motif 7 | LSFLKKNEGDTTSPIGPMEFNLPAGLNTNDFETAIDDEGLLTLTFKKLVP | 5 | 50 |
Motif 8 | MCLGNNPYQCSVFWYEDVNLVHFKYPLPPGAKQEDVKVEID | 7 | 41 |
Motif 9 | EEEKKPEVKAIEISG | 11 | 15 |
Motif 10 | QFPQETSAFVNTRVD | 9 | 15 |
Table 3 Conserved structural motifs built from the primary sequences
保守基序名称 Motif name | 氨基酸序列 Amino acid sequence | 起始位点 Site | 序列长度/aa Width |
---|---|---|---|
Motif 1 | WKETPEAHVFKADLPGLRKEEVKVE | 32 | 25 |
Motif 2 | KMDQIKASMENGVLTVTVPK | 33 | 20 |
Motif 3 | SSGMFSRRFRLPENA | 31 | 15 |
Motif 4 | VEDDRVLQISGQRKIEKEDKN | 27 | 21 |
Motif 5 | MSLIPSFFGNRRSSVFDPFSLDVWDPFRDF | 8 | 30 |
Motif 6 | DTWHRVER | 16 | 8 |
Motif 7 | LSFLKKNEGDTTSPIGPMEFNLPAGLNTNDFETAIDDEGLLTLTFKKLVP | 5 | 50 |
Motif 8 | MCLGNNPYQCSVFWYEDVNLVHFKYPLPPGAKQEDVKVEID | 7 | 41 |
Motif 9 | EEEKKPEVKAIEISG | 11 | 15 |
Motif 10 | QFPQETSAFVNTRVD | 9 | 15 |
Fig. 5 Expression analysis of HSP20 candidate genes in response to 105 CFU · mL-1 Xcc Different letters indicate significant differences(P < 0.05). The same below.
Fig. 8 Symptoms in resistance identification of sweet orange leaves A:Buffer control;B:HSP20 candidate genes overexpression vector Agrobacterium;C:Buffer + Xcc;D:HSP20 candidate genes overexpression vector agrobacterium + Xcc。1:CsHSP23.3;2:CmHSP23.3;3:CsHSP17.9;4:CmHSP17.9;5:CsHSP18.5;6:CmHSP18.5;P:pCAMIBA1300 vector control.
Fig. 9 Xcc quantitative analysis of overexpression of HSP20 candidate genes in sweet orange leaves Different letters indicate significant difference at 0.05 levels Xcc growth by buffer and overexpression of HSP20 candidate genes on the same day.
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