绿豆象热激蛋白超家族基因的鉴定及表达分析

doi:10.3864/j.issn.0578-1752.2023.19.009

Abstract

Abstract:

【Objective】The purpose of this study is to identify the gene members of the Callosobruchus chinensis heat shock protein (HSP) superfamily, and to clarify the expression changes of HSP genes in C. chinensis after high and low temperature stress, so as to provide a theoretical basis for further exploration of HSP gene function.【Method】The CDS and protein sequences of HSP genes of different insects were downloaded from Insect Base 2.0 and used as a reference for local BLASTp and tBLASTn comparison search in the full-length transcriptome sequencing database of C. chinensis. At the same time, target sequences were screened again by combining HMMER and key words to complete the summary of search results. Bioinformatics analysis of HSP superfamily genes in C. chinensis was performed using CDD, MEGA, ProtParam, and other online analytical tools. Seven candidate HSP genes were screened out based on high and low temperature transcriptome sequencing data of C. chinensis adults and the expression characteristics of 7 CcHsps were compared and analyzed by qRT-PCR technique under different developmental stages and temperature stresses of C. chinensis.【Result】A total of 31 HSP genes were identified, including 3 HSP90s, 8 HSP70s, 8 HSP60s, and 12 sHSPs (small HSP). Physicochemical analysis showed that the proteins encoded by CcHsps contain 159-776 amino acid residues (aa), the molecular weights are about 18.4-88.9 kDa, and the theoretical isoelectric points are 4.95-9.17. Subcellular localization results showed that most CcHsps were located in the cytoplasm, while a few genes were located in the mitochondrial matrix, endoplasmic reticulum and nucleus. Phylogenetic analysis showed that different family members of HSPs in C. chinensis could integrate well with HSP in other insects, which indicating their evolutionary conservation. The results of qRT-PCR showed that the 7 candidate CcHsps were differentially expressed under different temperature stresses. After high temperature stress, the expression level of CcHsp20.102 in male and female adults was up-regulated by 1 000 and 500 times, respectively, and the expression level of CcHsp70-5 in male and female adults was up-regulated by 500 and 450 times. After the larvae undergoing high and low temperature stress, the expression level of CcHsp19.855 and CcHsp70-5 was significantly different.【Conclusion】A total of 31 complete HSP superfamily gene members were identified by the full-length transcriptome sequencing data of C. chinensis, which were divided into 4 subfamilies. Different HSP families had different gene structures, protein conserved domains and gene expression characteristics. The differential expression of 7 candidate CcHsps in different developmental stages and under different temperature stresses indicated that they played different functions and roles. It is speculated that CcHsp20.102 and CcHsp70-5 may perform important functions in the adult resistance to high temperature stress, and the high temperature tolerance of larvae may be related to the differential expression of CcHsp19.855 and CcHsp70-5.

Key words: Callosobruchus chinensis, full-length transcriptome sequencing, heat shock protein, gene family, bioinformatics analysis, expression analysis

ZHANG Xin, YANG XingYu, ZHANG ChaoRan, ZHANG Chong, ZHENG HaiXia, ZHANG XianHong. Identification and Expression Analysis of Heat Shock Protein Superfamily Genes in Callosobruchus chinensis[J].Scientia Agricultura Sinica, 2023, 56(19): 3814-3828.

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URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2023.19.009

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Figures/Tables 8

Table 1

Table 2

Physicochemical properties of protein encoded by HSP superfamily gene in C. chinensis"

基因名 Gene name	编码数 CDS number	氨基酸数 AA number	分子量 MW (kDa)	等电点 PI	不稳定指数 II	脂肪指数 AI	亲水性平均值 GH	亚细胞定位 SL
CcHsp18.444	492	163	18.444	7.75	52.21	71.04	-0.942	细胞质Cytoplasm
CcHsp18.572	480	159	18.572	6.45	39.20	83.96	-0.686	细胞质、细胞核 Cytoplasm, nucleus
CcHsp19.121	504	167	19.121	8.79	53.82	73.41	-0.863	细胞质Cytoplasm
CcHsp19.855	525	174	19.855	6.96	43.56	78.39	-0.690	细胞质Cytoplasm
CcHsp20.102	531	176	20.102	6.97	48.14	76.42	-0.657	细胞质Cytoplasm
CcHsp20.103	534	177	20.103	6.30	59.44	84.24	-0.541	细胞质Cytoplasm
CcHsp20.956	533	176	20.956	6.24	62.18	77.68	-0.658	细胞质Cytoplasm
CcHsp21.032	573	190	21.032	7.59	56.39	67.58	-0.552	线粒体基质 Mitochondrial matrix
CcHsp21.936	585	194	21.936	8.34	57.23	85.82	-0.730	细胞质、细胞核 Cytoplasm, nucleus
CcHsp22.154	591	196	22.154	6.18	56.98	89.90	-0.610	细胞质Cytoplasm
CcHsp22.763	606	201	22.763	7.90	65.18	85.72	-0.544	细胞质Cytoplasm
CcHsp24.313	600	199	24.313	7.94	51.07	62.61	-0.957	细胞质Cytoplasm
CcHsp60-1	1605	534	57.326	6.36	33.89	111.59	0.078	细胞质Cytoplasm
CcHsp60-2	1605	534	57.594	5.74	35.65	101.78	-0.063	细胞核Nucleus
CcHsp60-3	1593	530	57.978	6.84	28.57	101.94	-0.122	细胞质Cytoplasm
CcHsp60-4	1626	541	58.777	5.84	40.52	104.21	-0.032	细胞质Cytoplasm
CcHsp60-5	1623	540	59.011	7.49	32.25	100.07	-0.089	细胞质Cytoplasm
CcHsp60-6	1593	530	60.038	6.74	32.45	104.41	-0.002	线粒体基质 Mitochondrial matrix
CcHsp60-7	1656	551	60.969	5.98	44.86	96.46	-0.230	细胞质Cytoplasm
CcHsp60-8	1725	574	61.300	5.44	33.69	95.96	-0.170	线粒体基质 Mitochondrial matrix
CcHsp70-1	1755	584	64.129	9.17	37.20	86.68	-0.346	细胞质Cytoplasm
CcHsp70-2	1767	588	64.938	5.66	21.45	89.20	-0.383	内质网 Endoplasmic reticulum
CcHsp70-3	1887	628	69.211	5.55	33.47	83.73	-0.429	细胞质Cytoplasm
CcHsp70-4	1905	634	69.864	4.97	25.51	84.89	-0.496	内质网 Endoplasmic reticulum
CcHsp70-5	1917	638	70.000	5.50	34.26	81.54	-0.467	细胞质Cytoplasm
CcHsp70-6	1935	644	70.496	5.50	38.54	82.87	-0.423	细胞质Cytoplasm
CcHsp70-7	1965	654	71.593	5.56	38.44	82.22	-0.445	细胞质Cytoplasm
CcHsp70-8	1980	659	72.901	5.07	25.59	86.56	-0.482	内质网Endoplasmic reticulum
CcHsp90-1	2091	696	78.856	8.04	40.02	88.97	-0.424	线粒体基质 Mitochondrial matrix
CcHsp90-2	2172	723	82.812	4.97	39.90	80.24	-0.681	细胞质Cytoplasm
CcHsp90-3	2331	776	88.900	4.95	39.29	81.40	-0.603	细胞外Extracellular

Table 2

Fig. 1

Fig. 2

Fig. 3

Table 3

Expression profiles of 31 CcHsps in high and low temperature transcriptomes of C. chinensis adult"

基因ID Gene ID	基因名 Gene name	描述 Description	A1 (FPKM)	A2 (FPKM)	A3 (FPKM)	B1 (FPKM)	B2 (FPKM)	B3 (FPKM)	C1 (FPKM)	C2 (FPKM)	C3 (FPKM)
F01_transcript_43299	CcHsp18.572	Alpha-crystallin B chain-like	8.98	5.94	4.90	5188.18	3905.53	4239.34	11.05	17.72	0
F01_transcript_42933	CcHsp19.855	Alpha-crystallin B chain-like	20.60	26.01	18.35	5155.01	5338.97	5536.08	49.24	27.53	19.29
F01_transcript_40250	CcHsp20.102	Protein lethal (2) essential for life	8.19	12.68	11.84	14663.45	14417.42	12560.95	12.78	42.34	10.79
F01_transcript_39920	CcHsp21.032	Alpha-crystallin B chain-like	0	0	0	1.49	1.16	0.32	0	0	0
F01_transcript_38328	CcHsp19.121	Alpha-crystallin B chain-like	31.83	30.27	19.40	793.80	422.20	553.81	18.71	42.25	15.67
F01_transcript_28534	CcHsp18.444	Alpha-crystallin B chain-like	0	2.17	1.50	12.66	0	0	0.14	0.65	0
F01_transcript_27941	CcHsp20.103	Protein lethal (2) essential for life	29.99	21.96	22.74	16.40	70.62	4.77	64.49	13.95	21.07
F01_transcript_24610	CcHsp22.154	Protein lethal (2) essential for life	26.12	27.01	16.63	1208.61	508.81	769.67	17.83	34.94	13.28
F01_transcript_24085	CcHsp20.956	Protein lethal (2) essential for life	122.65	191.95	87.92	3007.42	3623.59	4306.85	118.63	76.41	78.69
F01_transcript_24011	CcHsp22.763	Alpha-crystallin B chain-like	0	0	0	0	0	0	0	0	0
F01_transcript_22315	CcHsp21.936	Protein lethal (2) essential for life	20.37	49.74	23.53	616.41	1482.13	892.17	13.49	26.33	37.90
F01_transcript_5515	CcHsp24.313	Alpha-crystallin B chain-like	239.53	273.50	197.18	250.14	431.63	251.55	208.14	381.78	263.16
F01_transcript_128	CcHsp60-8	60 kDa heat shock protein	152.30	368.87	363.28	445.95	297.55	250.78	237.47	397.82	120.83
F01_transcript_40995	CcHsp60-5	T-complex protein 1 subunit eta-like	41.21	38.76	37.33	71.18	21.85	33.86	38.20	49.72	18.06
F01_transcript_20569	CcHsp60-4	T-complex protein 1 subunit epsilon	13.40	21.90	49.28	31.97	30.52	16.34	14.34	44.61	20.79
F01_transcript_694	CcHsp60-3	T-complex protein 1 subunit zeta	102.08	125.65	112.61	100.87	67.07	98.74	86.41	116.36	71.93
F01_transcript_639	CcHsp60-6	T-complex protein 1 subunit alpha	46.86	86.59	94.75	95.54	54.49	94.64	71.68	122.22	69.76
F01_transcript_578	CcHsp60-2	T-complex protein 1 subunit beta	63.48	71.65	69.67	66.85	61.50	77.62	63.93	95.76	70.86
F01_transcript_501	CcHsp60-1	T-complex protein 1 subunit delta	80.57	93.12	71.84	58.52	45.90	53.01	36.07	83.26	77.10
F01_transcript_442	CcHsp60-7	T-complex protein 1 subunit gamma	89.12	101.69	91.83	85.56	64.15	75.93	95.03	109.45	79.94
F01_transcript_73	CcHsp70-6	Heat shock protein 70	6.03	3.74	9.44	1609.09	1163.31	1709.61	6.05	12.11	0
F01_transcript_18358	CcHsp70-2	Heat shock 70 kDa protein cognate 3 isoform X1	426.14	211.25	179.19	536.20	53.68	35.18	105.66	82.65	21.55
F01_transcript_16333	CcHsp70-4	Heat shock 70 kDa protein cognate 3	0.31	0	0	0	0.77	0	0	0	0
F01_transcript_277	CcHsp70-3	Heat shock 70 kDa protein cognate 2	39.45	40.46	65.39	55.88	81.61	43.11	83.69	35.19	31.18
F01_transcript_168	CcHsp70-5	Heat shock protein 70	10.63	10.46	9.83	5992.26	4679.61	6348.31	11.82	21.93	5.07
F01_transcript_122	CcHsp70-8	Heat shock 70 kDa protein cognate 3 isoform X3	112.90	305.41	319.01	851.12	1148.55	644.20	274.10	555.56	385.90
F01_transcript_120	CcHsp70-1	Heat shock protein 70	0.03	1.12	2.54	133.45	574.24	386.62	1.94	1.71	1.50
F01_transcript_56	CcHsp70-7	Heat shock protein 70	0	110.53	0	0	2.09	0	0	1.63	1.13
F01_transcript_42638	CcHsp90-1	Heat shock protein 75 kDa	20.55	22.54	21.28	21.17	22.56	23.64	18.12	22.08	30.43
F01_transcript_59	CcHsp90-3	Endoplasmin	254.32	274.77	261.66	361.26	319.09	296.49	136.10	214.31	168.92
F01_transcript_57	CcHsp90-2	Heat shock protein 83	1122.57	1278.61	1487.83	3408.15	3540.80	3795.53	758.88	1352.60	1729.74

Table 3

Table 4

Statistical analysis of expression level of differentially expressed HSP gene under high and low temperature stress transcriptomes"

基因ID Gene ID	A1_Count	A2_Count	A3_Count	B1_Count	B2_Count	B3_Count	错误发现率 FDR	差异倍数的对数值 Log₂FC	基因调节 Gene regulated
F01_transcript_43299	83	52	47	45192	36760	34185	1.25E-281	8.875292037	Up
F01_transcript_38328	326	295	207	7703	4431	4996	6.02E-50	4.188931643	Up
F01_transcript_24624	63	152	42	22981	31416	26459	1.09E-69	6.883143797	Up
F01_transcript_3613	125	152	105	23298	32452	32178	3.91E-195	7.499200641	Up
F01_transcript_20561	0	0	0	54	42	36	6.81E-13	3.780936629	Up
F01_transcript_25418	23	7	18	19904	19924	16508	1.20E-208	9.246980188	Up
F01_transcript_5284	33	23	0	3143	1904	1905	0.019906364	1.64057389	Up
F01_transcript_36652	31	52	52	128	278	165	2.89E-05	1.884678396	Up
F01_transcript_43228	0	0	1	67	149	724	1.88E-08	3.227036338	Up
F01_transcript_33596	270	269	146	693	709	451	2.48E-05	1.476145857	Up
F01_transcript_41848	1258	1272	1250	3286	3617	2559	1.85E-13	1.448039781	Up
F01_transcript_28277	1	3	7	86	296	795	1.67E-09	3.378050236	Up
F01_transcript_6174	11	6	54	4940	9656	5025	7.99E-21	4.73690697	Up
F01_transcript_18613	0	12	4	5653	5050	3444	1.44E-65	7.225268629	Up
F01_transcript_17534	42	21	92	753	630	959	3.63E-11	3.117146973	Up
F01_transcript_42430	2164	1080	1190	7186	7757	5735	2.46E-12	2.200081315	Up
F01_transcript_38496	650	797	647	1878	2100	1393	1.56E-09	1.459606834	Up
F01_transcript_40250	53	77	79	88274	93445	68696	0	9.709356267	Up
F01_transcript_29133	1	7	7	1406	1481	1820	1.86E-85	1.723134458	Up
F01_transcript_19811	32	30	55	154	154	108	3.71E-05	1.065158394	Up
F01_transcript_47	296002	298628	475364	749443	723281	659223	0.003868698	1.109360769	Up
F01_transcript_34439	20	15	20	42	42	36	0.039086603	1.605846729	Up
F01_transcript_122	3620	9409	10704	26414	38741	19064	0.001900569	6.502458533	Up
F01_transcript_30785	0	0	0	423	320	442	1.43E-52	1.382236836	Up
F01_transcript_46898	164	175	56	253	693	263	0.02788211	4.184196848	Up
F01_transcript_22315	242	565	292	6981	18172	9444	2.10E-24	4.4489631	Up
F01_transcript_24085	1209	1799	902	27990	36460	37206	2.08E-50	1.286238774	Up
F01_transcript_25973	19	16	16	36	65	34	0.024418377	1.601501158	Up
F01_transcript_45548	90	137	174	425	381	428	1.92E-05	3.316756015	Up
F01_transcript_35814	3	7	3	82	82	60	6.56E-14	5.119419417	Up
F01_transcript_21658	17	14	17	1080	557	1040	5.07E-47	3.788445916	Up
F01_transcript_37597	601	727	663	11682	10180	6929	1.31E-55	1.272772815	Up
F01_transcript_28254	31	11	27	77	55	52	0.033458361	6.722645203	Up
F01_transcript_73	206	123	339	53457	42010	54200	1.57E-75	1.4927542	Up
F01_transcript_21952	22	11	52	133	87	98	0.021134189	1.513921055	Up
F01_transcript_57	38595	42246	53536	113486	128163	120611	7.83E-09	1.538732717	Up
F01_transcript_23965	425	382	379	927	1591	841	2.81E-06	7.802804908	Up
F01_transcript_42933	223	268	207	52943	59323	52969	0	2.20861966	Up
F01_transcript_27028	6	4	3	44	49	18	0.000136632	1.259693495	Up
F01_transcript_19334	197	244	249	536	451	555	1.31E-05	1.467376685	Up
F01_transcript_11744	43	21	0	2103	1367	1285	0.045002773	4.400867923	Up
F01_transcript_27257	1970	2940	2585	57913	52839	57508	1.67E-76	4.702861053	Up
F01_transcript_24610	349	344	231	15406	7026	9202	3.65E-48	1.199883723	Up
F01_transcript_33669	222	189	244	530	461	405	6.09E-06	8.919378418	Up
F01_transcript_168	325	306	314	177176	150364	178888	0	1.504407815	Up
F01_transcript_120	1	34	86	4186	19580	11567	0.036109917	6.953691731	Up
F01_transcript_23373	17	20	17	5167	4047	2431	3.95E-103	1.674610512	Up
F01_transcript_3052	452	458	419	1457	1384	1062	1.08E-18	5.053735916	Up
F01_transcript_41976	16	2	1	920	920	826	1.98E-27	3.220154002	Up
F01_transcript_4271	0	13	18	434	520	280	4.73E-09	1.781266067	Up
F01_transcript_36223	8	0	0	24988	25210	20569	0.009242401	8.875292037	Up

Table 4

Fig. 4

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基因名 Gene name	引物序列 Primer sequence (5′ to 3′)	产物长度 Product length (bp)	标准曲线 Standard curve (Y=)	扩增效率Amplification efficacy (%)	相关系数 Correlation coefficient
CcHsp90-2	F: TAATCAGTCGTTGAAGCAG R: TACATTGCTGTTGCGATTC	103	-3.276*log₂X+15.55	102.0	0.992
CcHsp70-5	F: ACAGATTCCGTGTTTTGA R: GCTAAAACTCAGTCTATTCG	109	-3.379*log₂X+14.43	97.7	0.987
CcHsp70-6	F: CGACAGTGTAAACAACTCTAGC R: GAATGTCACAGCAGATACTACTA	160	-3.184*log₂X+22.40	106.1	0.998
CcHsp60-3	F: GCATAATGGGTCTGCGTCG R: AGCATAGCCGAGGTTGGA	116	-3.178*log₂X+19.36	106.4	0.998
CcHsp20.102	F: TGCTAGTGTCAGTGTTCAAC R: ACATACAAAATCAACCTTCC	126	-3.146*log₂X+24.81	107.9	0.994
CcHsp19.855	F: GAGCGACTTTTCGTATCAA R: TCCAACACCAATCTTTCAT	175	-3.197*log₂X+20.01	105.5	0.999
CcHsp18.572	F: CGTTTTTTGACTGGAATAC R: TGGATAGAAATACCTGCTT	144	-3.362*log₂X+20.68	98.4	0.999
β-tubulin	F: CTTCAGAGGCAGGATGTC R: TCCCCTTGGTGGAATGTC	133	-3.269*log₂X+16.20	102.3	0.997

Identification and Expression Analysis of Heat Shock Protein Superfamily Genes in Callosobruchus chinensis

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