钙依赖蛋白激酶CPK17在花粉管生长中的功能分析

doi:10.7668/hbnxb.20194326

摘要/Abstract

摘要：

钙依赖蛋白激酶(CPK)是一类响应钙信号的蛋白激酶,参与调节植物繁殖中钙信号和细胞骨架精密控制的花粉管极性生长。为揭示拟南芥CPK在花粉管极性生长中的功能,利用生物信息学方法分析CPK家族成员的表达模式和进化关系,构建持续激活(CA)和显性抑制(DN)型CPK17突变体。基于基因枪技术,观察CA-CPK17和DN-CPK17过表达花粉管的表型变化,比较高钙和低钙胁迫对CPK17和CA-CPK17过表达花粉管生长的影响,分析CPK17与同源蛋白CPK34的共同表达情况。另外预测CPK17的互作蛋白网络,并对部分互作蛋白进行验证。结果显示,拟南芥中有7个花粉特异表达的CPKs,其中CPK17和CPK34具有高度的相似性(93%),并在成熟花粉中高表达,在其他组织中低表达或不表达。过量表达CPK17对花粉管生长没有显著影响,而CA-CPK17则导致花粉管去极化生长和顶端肿胀,DN-CPK17无明显效果。过量表达CPK34也引起类似的花粉管生长缺陷,而同时过表达CPK17和CPK34则使缺陷加重。钙胁迫试验显示,CPK17过表达花粉管对低钙条件不敏感,而对高钙条件敏感。而缺乏调控域的CA-CPK17过表达花粉管对高钙和低钙条件均不敏感。预测的CPK17互作蛋白主要是ROP负调节因子,但酵母双杂交试验未能证实存在互作关系。这些结果表明,CPK17的C端调控域在钙信号的响应中起重要作用。CPK17和CPK34在花粉管生长中具有协同作用,可能通过调控钙离子的内流,进而影响胞内的钙离子梯度。

关键词: 花粉管, 极性生长, 基因枪, 钙依赖蛋白激酶, 钙信号途径

Abstract:

Calcium-dependent protein kinases(CPK)are a class of protein kinases that respond to calcium signals and participate in regulating the polar growth of pollen tubes,which is crucial for plant reproduction and is precisely controlled by calcium signals and the cytoskeleton.To reveal the function of CPK in the polar growth of pollen tubes in Arabidopsis,bioinformatics methods were used to analyze the expression patterns and evolutionary relationships of CPK family members,and constitutively active(CA)and dominant-negative(DN)CPK17 mutants were constructed.Based on gene gun technology,we observed the phenotypic changes of CA-CPK17 and DN-CPK17 overexpressing pollen tubes,compared the effects of high and low calcium stress on the growth of CPK17 and CA-CPK17 overexpressing pollen tubes,and analyzed the co-expression of CPK17 and its homologous protein CPK34.Additionally,the protein interaction network of CPK17 was predicted,and some of the interacting proteins were validated.The results showed that there were seven pollen-specifically expressed CPKs in Arabidopsis thaliana.Among them,CPK17 and CPK34 had a high similarity (93%),and were highly expressed in mature pollen,and low or no expression in other tissues.Overexpression of CPK17 had no significant effect on pollen tube growth,while CA-CPK17 caused depolarized growth and tip swelling of pollen tubes,and DN-CPK17 had no obvious effect.Overexpression of CPK34 also caused similar defects in pollen tube growth,and co-overexpression of CPK17 and CPK34 aggravated the defects.Calcium stress experiments showed that overexpressing CPK17 pollen tubes were insensitive to low calcium conditions,but sensitive to high calcium conditions.While pollen tubes overexpressing CA-CPK17(lacking the regulatory domain)were insensitive to both high and low calcium conditions.The predicted interacting proteins of CPK17 were mainly ROP negative regulators,but yeast two-hybrid experiments failed to confirm the interaction.These results indicated that the C-terminal regulatory domain of CPK17 plays an important role in the response to calcium signals.CPK17 and CPK34 have synergistic effects on pollen tube growth,possibly by regulating calcium influx and affecting intracellular calcium gradient.

Key words: Pollen tube, Polar growth, Gene gun, Calcium-dependent protein kinase, Ca²⁺ signaling pathway

周利明, 任中慧, 刘格, 房玮. 钙依赖蛋白激酶CPK17在花粉管生长中的功能分析[J]. 华北农学报, 2024, 39(S1): 135-142. doi: 10.7668/hbnxb.20194326.

ZHOU Liming, REN Zhonghui, LIU Ge, FANG Wei. Functional Analysis of Calcium Dependent Protein Kinase 17 in Pollen Tube Growth[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 135-142. doi: 10.7668/hbnxb.20194326.

http://www.hbnxb.net/CN/Y2024/V39/IS1/135

图/表 8

图1 CPKs系统进化与组织表达热图

Fig.1 Heat map of CPKs system evolution and tissue expression

表1 CPK基因家族信息分析

Tab.1 Information analysis of CPK family

名称 Name	基因ID Gene ID	蛋白质长度/aa Protein length	染色体定位 Chromosome localization	高量表达组织 Highly expressed tissue	花粉表达占比/% Ratio of pollen expression
CPK1	At5g04870	610	Chr5:1416564-1420554	根和叶	0.02
CPK2	At3g10660	646	Chr3:3331064-3334986	花粉和根	58.53
CPK3	At4g23650	529	Chr4:12324758-12327651	根和叶	0.60
CPK4	At4g09570	501	Chr4:6049441-6052380	根和芽	14.48
CPK5	At4g35310	556	Chr4:16801605-16805005	根和叶	0.21
CPK6	At2g17290	544	Chr2:7516330-7519662	花粉和根	29.04
CPK7	At5g12480	535	Chr5:4047513-4050643	根和种子	1.76
CPK8	At5g19450	533	Chr5:6558331-6562057	根和种子	1.38
CPK9	At3g20410	541	Chr3:7115819-7119144	根和叶	0.06
CPK10	At1g18890	545	Chr1:6522764-6526075	叶和芽	0.03
CPK11	At1g35670	495	Chr1:13205345-13208289	花粉和根	38.25
CPK12	At5g23580	490	Chr5:7950202-7952619	花和果荚	<0.01
CPK13	At3g51850	528	Chr3:19232358-19235891	茎和花	0.28
CPK14	At2g41860	530	Chr2:17467282-17470157	花粉和花	95.30
CPK15	At4g21940	561	Chr4:11640735-11643762	花和叶	0.34
CPK16	At2g17890	571	Chr2:7769521-7772782	花粉和花	95.01
CPK17	At5g12180	528	Chr5:3936813-3939596	花粉和花	91.99
CPK18	At4g36070	561	Chr4:17056587-17059706	花粉和花	95.24
CPK19	At1g61950	551	Chr1:22899389-22902118	根和种子	<0.01
CPK20	At2g38910	583	Chr2:16244906-16247855	花粉和花	95.92
CPK21	At4g04720	531	Chr4:2393653-2397804	茎和芽	0.10
CPK22	At4g04710	500	Chr4:2390307-2392989	根和种子	<0.01
CPK23	At4g04740	533	Chr4:2402897-2408737	叶和芽	15.14
CPK24	At2g31500	582	Chr2:13413738-13416602	花粉和花	89.50
CPK25	At2g35890	520	Chr2:15066614-15069359	花粉和根	76.93
CPK26	At4g38230	514	Chr4:17928677-17931511	花粉和根	73.74
CPK27	At4g04700	485	Chr4:2385038-2388105	根和芽	0.17
CPK28	At5g66210	523	Chr5:26456142-26459654	芽和根	2.06
CPK29	At1g76040	561	Chr1:28537593-28540707	茎和根	0.11
CPK30	At1g74740	541	Chr1:28079946-28082671	根和茎	1.08
CPK31	At4g04695	484	Chr4:2381348-2384121	根和叶	<0.01
CPK32	At3g57530	538	Chr3:21296457-21299704	花粉和芽	40.29
CPK33	At1g50700	547	Chr1:18781764-18784582	果荚和花	<0.01
CPK34	At5g19360	523	Chr5:6521405-6523954	花粉和花	95.61

表1 CPK基因家族信息分析

Tab.1 Information analysis of CPK family

名称 Name	基因ID Gene ID	蛋白质长度/aa Protein length	染色体定位 Chromosome localization	高量表达组织 Highly expressed tissue	花粉表达占比/% Ratio of pollen expression
CPK1	At5g04870	610	Chr5:1416564-1420554	根和叶	0.02
CPK2	At3g10660	646	Chr3:3331064-3334986	花粉和根	58.53
CPK3	At4g23650	529	Chr4:12324758-12327651	根和叶	0.60
CPK4	At4g09570	501	Chr4:6049441-6052380	根和芽	14.48
CPK5	At4g35310	556	Chr4:16801605-16805005	根和叶	0.21
CPK6	At2g17290	544	Chr2:7516330-7519662	花粉和根	29.04
CPK7	At5g12480	535	Chr5:4047513-4050643	根和种子	1.76
CPK8	At5g19450	533	Chr5:6558331-6562057	根和种子	1.38
CPK9	At3g20410	541	Chr3:7115819-7119144	根和叶	0.06
CPK10	At1g18890	545	Chr1:6522764-6526075	叶和芽	0.03
CPK11	At1g35670	495	Chr1:13205345-13208289	花粉和根	38.25
CPK12	At5g23580	490	Chr5:7950202-7952619	花和果荚	<0.01
CPK13	At3g51850	528	Chr3:19232358-19235891	茎和花	0.28
CPK14	At2g41860	530	Chr2:17467282-17470157	花粉和花	95.30
CPK15	At4g21940	561	Chr4:11640735-11643762	花和叶	0.34
CPK16	At2g17890	571	Chr2:7769521-7772782	花粉和花	95.01
CPK17	At5g12180	528	Chr5:3936813-3939596	花粉和花	91.99
CPK18	At4g36070	561	Chr4:17056587-17059706	花粉和花	95.24
CPK19	At1g61950	551	Chr1:22899389-22902118	根和种子	<0.01
CPK20	At2g38910	583	Chr2:16244906-16247855	花粉和花	95.92
CPK21	At4g04720	531	Chr4:2393653-2397804	茎和芽	0.10
CPK22	At4g04710	500	Chr4:2390307-2392989	根和种子	<0.01
CPK23	At4g04740	533	Chr4:2402897-2408737	叶和芽	15.14
CPK24	At2g31500	582	Chr2:13413738-13416602	花粉和花	89.50
CPK25	At2g35890	520	Chr2:15066614-15069359	花粉和根	76.93
CPK26	At4g38230	514	Chr4:17928677-17931511	花粉和根	73.74
CPK27	At4g04700	485	Chr4:2385038-2388105	根和芽	0.17
CPK28	At5g66210	523	Chr5:26456142-26459654	芽和根	2.06
CPK29	At1g76040	561	Chr1:28537593-28540707	茎和根	0.11
CPK30	At1g74740	541	Chr1:28079946-28082671	根和茎	1.08
CPK31	At4g04695	484	Chr4:2381348-2384121	根和叶	<0.01
CPK32	At3g57530	538	Chr3:21296457-21299704	花粉和芽	40.29
CPK33	At1g50700	547	Chr1:18781764-18784582	果荚和花	<0.01
CPK34	At5g19360	523	Chr5:6521405-6523954	花粉和花	95.61

图2 组成激活型CPK17(CA-CPK17)与显著抑制型CPK17(DN-CPK17)的构建图解

Fig.2 Construction diagram of constitutively active CPK17(CA-CPK17)and dominant-negative CPK17(DN-CPK17)

图3 CA-和DN-CPK17在花粉管中的表型分析 A.CPK17、CA-CPK17和DN-CPK17过量表达的花粉管典型图像,标尺=30 μm;B.CA-CPK17和DN-CPK17过量表达引起的花粉管生长缺陷的定量分析,数据表示为均值±标准差,n=40~60。不同字母表示差异显著(P<0.05)。图4-5同。

Fig.3 Phenotypic analysis of CA-and DN-CPK17 in pollen tubes A.Typical images of pollen tubes overexpressing CPK17,CA-CPK17 and DN-CPK17.Scale bar = 30 μm;B.Quantitative analysis of pollen tube growth defects caused by overexpression of CA-CPK17 and DN-CPK17.Data are presented as mean ± standard deviation,n=40-60. Different letters indicate significant differences (P<0.05). The same as Fig.4-5.

图4 钙胁迫对花粉管极性生长的影响花粉管长度以均值±标准差表示,n=20~30。相对变化率为花粉管长度在不同条件下与100 μmol/L条件下的比值,用百分比表示。

Fig.4 The effect of calcium stress on pollen tube polar growth Pollen tube length is shown as mean±standard deviation,n=20-30.Relative change rate is the ratio of pollen tube length under different conditions to that under 100 μmol/L condition,indicated as percentage.

图5 CPK17和CPK34共同表达花粉管的表型分析 A.CPK17和CPK34单独及共同过量表达的花粉管典型图像,标尺=30 μm;B.CPK17和CPK34单独及共同过量表达引起的花粉管生长缺陷的定量分析。数据表示为均值±标准差,n=40~60。

Fig.5 Phenotypic analysis of pollen tubes co-expressing CPK17 and CPK34 A.Typical images of pollen tubes overexpressing CPK17 and CPK34 individually or together.Scale bar=30 μm;B.Quantitative analysis of pollen tube growth defects caused by overexpression of CPK17 and CPK34 individually or together.Data are presented as mean±standard deviation,n=40-60.

图6 CPK17的互作蛋白网络图

Fig.6 CPK17 protein interaction network

图7 CPK17和ROP1负调节因子的酵母双杂交试验

Fig.7 Yeast two-hybrid assay between CPK17 and ROP1 negative regulators

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