柑橘HSP20家族基因鉴定及其响应溃疡病菌侵染表达分析

doi:10.16420/j.issn.0513-353x.2021-0331

摘要/Abstract

摘要：

为探究HSP20在柑橘（Citrus L.）响应溃疡病菌（Xanthomonas citri subsp. citri,Xcc）侵染过程中发挥的作用,鉴定CsHSP20家族基因。生物学信息分析显示共有42个,分为11个亚族,分布在9条染色体上,编码135 ~ 373个氨基酸,蛋白分子量15.17 ~ 41.71 kD,等电点4.53 ~ 10.07,92.9%的基因没有或只有1个内含子,各亚族基因间有高度相似的结构和保守基序,每个基因的启动子都具有与激素或胁迫相关的顺式作用元件。其次,对感病的冰糖橙和抗病的枸橼C-05进行接种Xcc和40 ℃热胁迫处理,qRT-PCR结果显示CsHSP20家族中的HSP17.9、HSP23.3、HSP18.5和HSP18.0均上调表达,但抗感材料之间差异明显,HSP23.3在枸橼C-05中受Xcc上调表达水平极显著,该结果与受Xcc侵染的转录组结果一致。在冰糖橙叶片瞬时超表达候选基因,Xcc定量分析结果显示枸橼C-05的HSP23.3抑制Xcc繁殖速度更明显。

关键词: 柑橘, 枸橼, HSP20, 溃疡病, 表达分析

Abstract:

Citrus canker,caused by is a bacterial disease,which severely influence citrus industry,and its pathogenicity is significantly enhanced under higher temperature and humidity environmental condition. HSP20 genes are abundant and highly conserved in higher plants,they play vital defense roles when plants are under biotic or abiotic stress. In order to investigate the role of HSP20 genes in response to Xanthomonas citri subsp. citri infection,the genes of HSP20 family in Citrus sinensis were identified. The analysis of biological information showed that there are 42 members,which can be divided into 11 subfamilies,they encode 135-373 aa and are distributed on nine chromosomes,their protein molecular weight are from 15.17 to 41.71 kD,isoelectric point are from 4.53 to 10.07,almost 92.9% of them not contain any intron or only one,and each subfamily share highly similar structures and conserved motifs,their promoter also has cis-acting elements response to hormones or stress. In order to access the role of CsHSP20 in response to Xcc and abiotic stress in resistant germplasm Citron C-05 with susceptible sweet orange,we have injected Xcc into the leaves of Citron C-05 and conducted a series of heat stress with small Citron C-05 plants. The relative expression levels of CsHSP20 have been screened by qRT-PCR,the results showed that HSP17.9,HSP23.3,HSP18.5 and HSP18.0 were up-regulated in both Xcc and heat stress,but their relative expression level in Citron C-05 is notably different from that of sweet orange under Xcc infection,especially HSP23.3. Interestingly,the induced expression patterns are consistent with the result of the transcriptomic analysis infected by Xcc. Then,these genes have been transiently overexpressed in sweet orange leaves,and Xcc quantitative analysis of Citron C-05 HSP23.3 showed that Xcc reproduction was inhibited.

Key words: citrus, citron, HSP20, canker, expression analysis

中图分类号:

S666

张凯, 麻明英, 王萍, 李益, 金燕, 盛玲, 邓子牛, 马先锋. 柑橘HSP20家族基因鉴定及其响应溃疡病菌侵染表达分析[J]. 园艺学报, 2022, 49(6): 1213-1232.

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.

图/表 12

参考文献 68

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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

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

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

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