白三叶几丁质酶基因家族鉴定及TrChit3功能分析

doi:10.11686/cyxb2024424

摘要/Abstract

摘要：

几丁质酶是一种糖苷水解酶，在植物生长发育过程中发挥着重要作用。目前几丁质酶在白三叶应答各种生物和非生物胁迫的相关研究报道相对较少。本研究对白三叶的几丁质酶基因家族进行了全基因组鉴定，得到了44个几丁质酶基因，不均匀分布于白三叶16条染色体上，通过系统进化分析将这些几丁质酶分为GH18和GH19分支，5个不同的小组：I~V。其中29个基因属于GH18亚家族，15个基因属于GH19亚家族，且同一亚族基因具有相似的结构和功能，启动子分析发现，这44个几丁质酶基因的启动子区域包含与生长发育、激素及逆境胁迫相关的多种顺式作用元件，此外，qRT-PCR分析显示，盐胁迫和干旱胁迫处理显著诱导了几丁质酶基因的表达，进一步验证了其在植物胁迫响应中的作用。选择TrChit3基因深入研究几丁质酶基因的抗旱能力。在拟南芥中过表达TrChit3基因并进行干旱胁迫处理，测定相对电导率、丙二醛等生理指标，结果显示在干旱条件下过表达TrChit3基因能够显著提高拟南芥的耐旱能力。研究结果为植物中几丁质酶基因的探索奠定了基础。

关键词: 白三叶, 几丁质酶, 基因家族, 表达分析

Abstract:

Chitinases are glycoside hydrolases that play an important role in plant growth and development. Relatively few studies have focused on the responses of chitinases to various biotic and abiotic stresses in white clover （Trifolium repens）. Therefore， in this study， we searched the whole genome of white clover to identify and characterize its chitinase gene family. A total of 44 chitinase genes were identified in the whole genome of white clover， and they were unevenly distributed on 16 chromosomes. Phylogenetic analysis of these chitinase genes classified them into two mega-groups （GH18 and GH19）， with five separate clusters （I-V）. Twenty-nine chitinase genes belonged to the GH18 subfamily and 15 belonged to the GH19 subfamily. The genes within the same subfamily genes encoded proteins with similar structures and functions. Promoter analysis showed that the promoter regions of these 44 chitinase genes contained a variety of cis-acting elements related to growth and development， hormones， and stress. Furthermore， quantitative reverse-transcription polymerase chain reaction analyses showed that salt stress and drought stress resulted in significant increases in the transcript levels of chitinase genes， which further verified their roles in the plant stress response. TrChit3 was selected for a detailed analysis of its role in drought resistance. Overexpression of TrChit3 in Arabidopsis thaliana enhanced its resistance to drought stress， as measured by changes in physiological indexes such as relative conductivity and malondialdehyde content. These results lay a foundation for further studies on the roles of chitinases in plants.

Key words: white clover, chitinase, gene family, expression analysis

秦晓芳, 何芷睿, 贾彤, 杨玉娇, 付薇, 李航, 彭燕. 白三叶几丁质酶基因家族鉴定及TrChit3功能分析[J]. 草业学报, 2025, 34(10): 187-201.

Xiao-fang QIN, Zhi-rui HE, Tong JIA, Yu-jiao YANG, Wei FU, Hang LI, Yan PENG. Identification of the chitinase gene family and functional analysis of TrChit3 from white clover[J]. Acta Prataculturae Sinica, 2025, 34(10): 187-201.

图/表 13

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基因ID Gene ID	上游引物Forward primer （5′-3′）	下游引物Reverse primer （5′-3′）
gene-P8452_04698	CCGAACCCCTAAATTGAAC	CCACCGATAGAAAGCAACA
gene-P8452_70179	TCAAATTATGTCAATCCAAAGG	GAGTTTCCGCCTAAGAAGTTAT
gene-P8452_47669	AGCCCAAGAGTTAGGGTTTAC	ACAAGCAGGTTCTGATGATGT
gene-P8452_10179	CATTGATCCTTTTGTTGGTCT	GTACCATCTCCTAAGTTTTGGC
gene-P8452_42935	TTCTTGACGGCATTGATTT	GCATTTCCTATCCAAGCATC
gene-P8452_00576	CATCGGAGGAGGTAATAGCA	AAGTCAACACCATCAAACCC
gene-P8452_23770	CAGGGACAGATTGGTATGG	TTGAGAAAGAGCCCAGTTG
gene-P8452_72355	TATTCTCTTTCATCGCCTCA	TGTTCATTTCGTTTGTGGTT
gene-P8452_10178	AGTCCACAATCACAAAAGAAAA	TCCAAACATAATCAAATAAACCA
gene-P8452_10182	TAGGTGATGCTGTTTTAGATGG	TTTGCTTTTGTGATTGTGAACT

基因ID Gene ID	上游引物Forward primer （5′-3′）	下游引物Reverse primer （5′-3′）
gene-P8452_04698	CCGAACCCCTAAATTGAAC	CCACCGATAGAAAGCAACA
gene-P8452_70179	TCAAATTATGTCAATCCAAAGG	GAGTTTCCGCCTAAGAAGTTAT
gene-P8452_47669	AGCCCAAGAGTTAGGGTTTAC	ACAAGCAGGTTCTGATGATGT
gene-P8452_10179	CATTGATCCTTTTGTTGGTCT	GTACCATCTCCTAAGTTTTGGC
gene-P8452_42935	TTCTTGACGGCATTGATTT	GCATTTCCTATCCAAGCATC
gene-P8452_00576	CATCGGAGGAGGTAATAGCA	AAGTCAACACCATCAAACCC
gene-P8452_23770	CAGGGACAGATTGGTATGG	TTGAGAAAGAGCCCAGTTG
gene-P8452_72355	TATTCTCTTTCATCGCCTCA	TGTTCATTTCGTTTGTGGTT
gene-P8452_10178	AGTCCACAATCACAAAAGAAAA	TCCAAACATAATCAAATAAACCA
gene-P8452_10182	TAGGTGATGCTGTTTTAGATGG	TTTGCTTTTGTGATTGTGAACT

基因ID Gene ID	氨基酸长度Aminoacids length （aa）	分子量Molecular weight （Da）	等电点Isoelectric point （PI）	不稳定指数 Instability index	脂肪族氨基酸指数 Aliphatic index	平均疏水性Grand average of hydrophobicity
gene-P8452_04693	298	32361.98	9.16	34.30	86.74	0.009
gene-P8452_04697	362	39842.48	9.26	34.73	86.22	-0.050
gene-P8452_04698	291	32409.61	5.60	40.36	87.22	-0.105
gene-P8452_04688	275	29055.34	4.37	31.32	82.04	-0.011
gene-P8452_00576	383	43275.95	8.33	31.26	78.69	-0.205
gene-P8452_04690	278	29609.18	5.15	34.46	86.44	0.059
gene-P8452_06296	383	43255.08	8.80	29.62	79.48	-0.199
gene-P8452_10187	297	32149.64	8.96	32.32	84.07	0.028
gene-P8452_10182	275	28960.20	4.41	33.27	82.76	-0.002
gene-P8452_10188	292	32297.52	5.74	37.78	86.61	-0.063
gene-P8452_10178	297	31928.65	5.02	35.58	82.83	-0.142
gene-P8452_10179	206	22738.68	5.11	31.38	87.14	-0.035
gene-P8452_13386	207	22795.73	5.11	30.86	86.71	-0.037
gene-P8452_18384	303	33888.14	6.93	42.01	82.38	-0.194
gene-P8452_17128	621	68415.97	5.22	28.83	92.17	0.005
gene-P8452_23770	365	39463.67	4.55	28.88	75.51	-0.168
gene-P8452_28219	132	14603.66	6.25	32.11	72.50	-0.164
gene-P8452_29322	362	39410.05	8.96	33.69	74.56	-0.281
gene-P8452_33777	331	35611.74	5.37	34.50	56.40	-0.303
gene-P8452_39032	256	27187.40	7.44	46.42	56.52	-0.245
gene-P8452_42935	294	31613.91	6.70	33.99	85.37	-0.010
gene-P8452_47669	294	31587.88	6.70	33.02	86.36	0.009
gene-P8452_53931	212	22708.93	4.37	28.04	61.75	-0.321
gene-P8452_53932	211	22945.46	5.46	31.39	57.82	-0.280
gene-P8452_53933	186	20238.60	4.99	28.93	60.91	-0.175
gene-P8452_53934	283	30502.81	4.71	32.67	59.36	-0.297
gene-P8452_53930	275	29591.09	6.83	36.52	62.22	-0.135
gene-P8452_53929	278	29839.26	5.27	31.46	59.78	-0.204
gene-P8452_54091	411	45795.84	5.50	47.07	87.83	-0.215
gene-P8452_58401	391	43516.31	5.45	45.42	90.77	-0.177
gene-P8452_58243	284	30703.01	4.66	33.45	58.10	-0.322
gene-P8452_58237	235	25189.95	4.57	35.09	67.36	-0.093
gene-P8452_58242	178	19133.09	4.55	21.51	65.84	-0.152
gene-P8452_58236	241	25954.70	4.81	31.04	64.85	-0.166
gene-P8452_63358	328	36123.93	9.07	42.80	80.49	-0.256
gene-P8452_59011	322	35664.54	6.23	24.44	73.63	-0.159
gene-P8452_68078	303	33447.00	8.72	35.38	82.61	-0.176
gene-P8452_63938	322	35664.54	6.23	24.44	73.63	-0.159
gene-P8452_70179	298	32589.35	6.71	23.03	96.54	0.063
gene-P8452_70182	297	31644.73	5.32	32.97	79.93	-0.004
gene-P8452_72355	378	41722.57	6.11	28.83	84.37	0.011
gene-P8452_75297	296	32327.00	6.13	24.14	95.57	0.034
gene-P8452_75301	270	28454.16	5.26	34.06	86.48	0.111
gene-P8452_77241	388	42619.43	6.11	33.05	82.71	-0.002

基因ID Gene ID	氨基酸长度Aminoacids length （aa）	分子量Molecular weight （Da）	等电点Isoelectric point （PI）	不稳定指数 Instability index	脂肪族氨基酸指数 Aliphatic index	平均疏水性Grand average of hydrophobicity
gene-P8452_04693	298	32361.98	9.16	34.30	86.74	0.009
gene-P8452_04697	362	39842.48	9.26	34.73	86.22	-0.050
gene-P8452_04698	291	32409.61	5.60	40.36	87.22	-0.105
gene-P8452_04688	275	29055.34	4.37	31.32	82.04	-0.011
gene-P8452_00576	383	43275.95	8.33	31.26	78.69	-0.205
gene-P8452_04690	278	29609.18	5.15	34.46	86.44	0.059
gene-P8452_06296	383	43255.08	8.80	29.62	79.48	-0.199
gene-P8452_10187	297	32149.64	8.96	32.32	84.07	0.028
gene-P8452_10182	275	28960.20	4.41	33.27	82.76	-0.002
gene-P8452_10188	292	32297.52	5.74	37.78	86.61	-0.063
gene-P8452_10178	297	31928.65	5.02	35.58	82.83	-0.142
gene-P8452_10179	206	22738.68	5.11	31.38	87.14	-0.035
gene-P8452_13386	207	22795.73	5.11	30.86	86.71	-0.037
gene-P8452_18384	303	33888.14	6.93	42.01	82.38	-0.194
gene-P8452_17128	621	68415.97	5.22	28.83	92.17	0.005
gene-P8452_23770	365	39463.67	4.55	28.88	75.51	-0.168
gene-P8452_28219	132	14603.66	6.25	32.11	72.50	-0.164
gene-P8452_29322	362	39410.05	8.96	33.69	74.56	-0.281
gene-P8452_33777	331	35611.74	5.37	34.50	56.40	-0.303
gene-P8452_39032	256	27187.40	7.44	46.42	56.52	-0.245
gene-P8452_42935	294	31613.91	6.70	33.99	85.37	-0.010
gene-P8452_47669	294	31587.88	6.70	33.02	86.36	0.009
gene-P8452_53931	212	22708.93	4.37	28.04	61.75	-0.321
gene-P8452_53932	211	22945.46	5.46	31.39	57.82	-0.280
gene-P8452_53933	186	20238.60	4.99	28.93	60.91	-0.175
gene-P8452_53934	283	30502.81	4.71	32.67	59.36	-0.297
gene-P8452_53930	275	29591.09	6.83	36.52	62.22	-0.135
gene-P8452_53929	278	29839.26	5.27	31.46	59.78	-0.204
gene-P8452_54091	411	45795.84	5.50	47.07	87.83	-0.215
gene-P8452_58401	391	43516.31	5.45	45.42	90.77	-0.177
gene-P8452_58243	284	30703.01	4.66	33.45	58.10	-0.322
gene-P8452_58237	235	25189.95	4.57	35.09	67.36	-0.093
gene-P8452_58242	178	19133.09	4.55	21.51	65.84	-0.152
gene-P8452_58236	241	25954.70	4.81	31.04	64.85	-0.166
gene-P8452_63358	328	36123.93	9.07	42.80	80.49	-0.256
gene-P8452_59011	322	35664.54	6.23	24.44	73.63	-0.159
gene-P8452_68078	303	33447.00	8.72	35.38	82.61	-0.176
gene-P8452_63938	322	35664.54	6.23	24.44	73.63	-0.159
gene-P8452_70179	298	32589.35	6.71	23.03	96.54	0.063
gene-P8452_70182	297	31644.73	5.32	32.97	79.93	-0.004
gene-P8452_72355	378	41722.57	6.11	28.83	84.37	0.011
gene-P8452_75297	296	32327.00	6.13	24.14	95.57	0.034
gene-P8452_75301	270	28454.16	5.26	34.06	86.48	0.111
gene-P8452_77241	388	42619.43	6.11	33.05	82.71	-0.002