玉米苯丙氨酸解氨酶（PAL）家族基因分析及ZmPAL5的功能研究

doi:10.3864/j.issn.0578-1752.2024.12.001

摘要/Abstract

摘要：

【目的】挖掘玉米抗旱关键基因、揭示其抗旱分子机制，为培育抗旱玉米新品种提供基因资源和理论指导。【方法】采用转录组数据结合加权基因共表达网络（weighted gene co-expression network，WGCNA）与抗旱性生理生化指标的筛选方法，鉴定与抗旱和复水相关的ZmPAL基因。利用生物信息学方法对编码PAL的基因进行全基因组分析。运用实时荧光定量PCR（quantitative real-time polymerase chain reaction，qRT-PCR）检测ZmPAL基因在干旱处理条件下的表达情况，以及ZmPAL5在不同自交系间的表达特性和在不同组织中的表达模式。最后，利用遗传转化分析ZmPAL5在玉米中的抗旱功能，并借助CRISPR/Cas9技术对PAL5同源基因进行缺失型拟南芥突变体的抗旱性分析。【结果】鉴定了19个玉米ZmPAL基因，其中6个基因聚集在第5染色体上，其编码蛋白多为亲水性酸性蛋白，且PAL家族基因的进化相对保守。ZmPAL基因启动子区域含有大量与激素和非生物应激响应相关的顺式作用元件。确定了6个核心基因，其中4个基因在干旱处理后显著上调表达。尤其是ZmPAL5在干旱胁迫后表达量增加了8.57倍。在干旱胁迫和复水处理条件下，发现抗旱自交系郑8713中ZmPAL5的表达水平显著高于旱敏感自交系B73。同时，ZmPAL5是一个组成型表达基因，在幼茎中表现出高水平的表达。过表达ZmPAL5玉米在干旱胁迫下生长良好，其相对含水量、木质素、叶绿素、可溶性蛋白、脯氨酸含量，以及超氧化物歧化酶、过氧化物酶、过氧化氢酶和抗坏血酸过氧化物酶的活性分别是野生型的1.52、1.49、1.47、1.43、1.44、1.41、1.53、1.41和1.35倍，但丙二醛含量是野生型的0.65倍。PAL5缺失型拟南芥突变体对干旱敏感。在干旱胁迫下，其生理生化指标与过表达ZmPAL5玉米的指标变化趋势相反。【结论】筛选出6个响应干旱胁迫的核心基因（ZmPAL3、ZmPAL5、ZmPAL6、ZmPAL8、ZmPAL11和ZmPAL13），其中，ZmPAL5的表达量与抗旱性呈正相关。ZmPAL5通过影响渗透调节物质含量和抗氧化酶活性正向调节植物的抗旱性和恢复能力。

关键词: 玉米, 苯丙氨酸解氨酶, ZmPAL5, 抗旱性, 生理生化

Abstract:

【Objective】 Mining the key drought-resistant genes of maize, revealing its drought-resistant molecular mechanism, and providing genetic resources and theoretical guidance for the cultivation of new drought-resistant maize varieties.【Method】Transcriptome data combined with weighted gene co-expression network (WGCNA) and screening methods for physiological and biochemical indicators of drought resistance were used to identify ZmPAL genes associated with drought resistance and rewatering. Genome-wide analysis of the genes encoding PAL was performed using bioinformatics methods. Quantitative real-time fluorescence PCR (qRT-PCR) was used to detect the expression of ZmPAL genes under drought treatment conditions, as well as the expression characteristics of ZmPAL5 among different inbred lines and the expression patterns in different tissues. Finally, genetic transformation was used to analyze the drought resistance function of ZmPAL5 in maize, and the deletion-type Arabidopsis mutant was analyzed for drought resistance with the help of CRISPR/Cas9 technology for the PAL5 homologous gene.【Result】Nineteen maize ZmPAL genes were identified, six of which were clustered on chromosome 5 and encoded proteins that were mostly hydrophilic acidic proteins and relatively evolutionarily conserved in the PAL family of genes. The promoter region of ZmPAL genes contained a large number of cis-acting elements associated with hormonal and abiotic stress responses. Six core genes were identified, four of which were significantly up-regulated for expression after drought treatment. In particular, ZmPAL5 showed an 8.57-fold increase in expression after drought stress. The expression level of ZmPAL5 was found to be significantly higher in the drought-resistant inbred line Zheng 8713 than in the drought-sensitive inbred line B73 under both drought stress and rewatering treatments. Meanwhile, ZmPAL5, a constitutively expressed gene, showed a high level of expression in young stems. Overexpressed ZmPAL5 maize grew well under drought stress, and its relative water content, lignin, chlorophyll, soluble protein, proline content, and activities of superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase were 1.52, 1.49, 1.47, 1.43, 1.44, 1.41, 1.53, 1.41, and 1.35 times, but the malondialdehyde content was 0.65 times that of the wild type. The PAL5-deficient Arabidopsis mutant was sensitive to drought. Under drought stress, its physiological and biochemical indexes showed the opposite trend to those of overexpression of ZmPAL5 maize. 【Conclusion】 Six core genes (ZmPAL3, ZmPAL5, ZmPAL6, ZmPAL8, ZmPAL11, and ZmPAL13) were screened in response to drought stress, in which the expression of ZmPAL5 was positively correlated with drought resistance. ZmPAL5 positively regulated the drought resistance and resilience of the plant by influencing the content of osmotically regulated substances and antioxidant enzyme activities.

Key words: maize, phenylalanine ammonia-lyase, ZmPAL5, drought resistance, physiology and biochemistry

曹丽茹, 叶飞宇, 库丽霞, 马晨晨, 庞芸芸, 梁小菡, 张新, 鲁晓民. 玉米苯丙氨酸解氨酶（PAL）家族基因分析及ZmPAL5的功能研究[J]. 中国农业科学, 2024, 57(12): 2265-2281.

CAO LiRu, YE FeiYu, KU LiXia, MA ChenChen, PANG YunYun, LIANG XiaoHan, ZHANG Xin, LU XiaoMin. Analysis of Maize Phenylalanine Ammonia-Lyase (PAL) Family Genes and Functional Study of ZmPAL5[J]. Scientia Agricultura Sinica, 2024, 57(12): 2265-2281.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.12.001

https://www.chinaagrisci.com/CN/Y2024/V57/I12/2265

图/表 8

表1

图1

表2

玉米ZmPAL蛋白的理化性质分析及亚细胞定位预测"

基因名称 Gene name	基因ID Gene ID	物理位置 Location (bp)	长度 Length (aa)	分子量 Molecular weight (kDa)	等电点 pI	亲水性系数 Grand average of hydropathicity	亚细胞定位预测 Prediction of subcellular localization
ZmPAL1	Zm00001d029015	Chr.1:54941440-54943661	398	43.19	6.52	-0.033	细胞质，叶绿体 Cytoplasmic, Chloroplast
ZmPAL2	Zm00001d029648	Chr.1:81308232-81312047	848	95.13	5.82	-0.293	叶绿体Chloroplast
ZmPAL3	Zm00001d031071	Chr.1:175969709-175971563	386	43.31	9.05	-0.214	叶绿体，线粒体 Chloroplast, Mitochondrial
ZmPAL4	Zm00001d033286	Chr.1:257622515-257624760	698	75.66	5.85	-0.04	质膜Plasma membrane
ZmPAL5	Zm00001d003015	Chr.2:29467931-29470598	715	76.78	5.63	-0.079	叶绿体，质膜，内质网 Chloroplast, Plasma membrane, Endoplasmic reticulum
ZmPAL6	Zm00001d003016	Chr.2:29538173-29541434	884	96.71	5.99	-0.195	质膜，叶绿体，线粒体 Plasma membrane, Chloroplast, Mitochondrial
ZmPAL7	Zm00001d048874	Chr.4:6825874-6829872	778	87.89	5.30	-0.296	细胞质Cytoplasmic
ZmPAL8	Zm00001d051161	Chr.4:146706971-146710589	557	60.43	6.02	-0.055	叶绿体Chloroplast
ZmPAL9	Zm00001d051163	Chr.4:146793740-146798809	718	77.43	6.12	-0.121	叶绿体，内质网 Chloroplast, Endoplasmic reticulum
ZmPAL10	Zm00001d051166	Chr.4:146846109-146848259	775	84.54	5.37	-0.163	叶绿体，质膜，细胞核 Chloroplast, Plasma membrane, Nuclear
ZmPAL11	Zm00001d013534	Chr.5:13763873-13782041	187	20.48	7.64	-0.441	叶绿体，线粒体 Chloroplast, Mitochondrial
ZmPAL12	Zm00001d017274	Chr.5:191418711-191422345	703	75.46	5.96	-0.069	叶绿体，质膜，内质网 Chloroplast, Plasma membrane, Endoplasmic reticulum
ZmPAL13	Zm00001d017275	Chr.5:191467430-191471500	719	77.4	6.20	-0.100	叶绿体，内质网 Chloroplast, Endoplasmic reticulum
ZmPAL14	Zm00001d017276	Chr.5:191474696-191476810	704	75.84	5.89	-0.066	叶绿体，内质网，细胞核 Chloroplast, Endoplasmic reticulum, Nuclear
ZmPAL15	Zm00001d017279	Chr.5:191539072-191541219	715	76.93	5.89	-0.083	叶绿体，质膜，细胞核 Chloroplast, Plasma membrane, Nuclear
ZmPAL16	Zm00001d017617	Chr.5:201803815-201807716	87	9.29	5.25	-0.266	叶绿体Chloroplast
ZmPAL17	Zm00001d021368	Chr.7:150079651-150084317	321	36.34	9.51	-0.302	叶绿体Chloroplast
ZmPAL18	Zm00001d023655	Chr.10:13756073-13759615	366	40.32	9.02	0.296	叶绿体，质膜，液泡 Chloroplast, Plasma membrane, Vacuolar
ZmPAL19	Zm00001d024512	Chr.10:75069687-75075183	727	81.54	5.32	-0.227	细胞核Nuclear

表2

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引物名称Primer name	引物序列Primer sequence (5′-3′)	用途Purpose
ZmPAL3-F	TCGGATTCACTGGCTTGCTG	检测基因在干旱胁迫下的表达量 Detection of gene expression under drought stress
ZmPAL3-R	GAGGATTGGACCGAAGTGGTA
ZmPAL5-F	ATGAACGGCACCGACAGCTA
ZmPAL5-R	CAGGGTGTTGATGCGGACGA
ZmPAL6-F	AGTGCTAGTGCTGCTACCCT
ZmPAL6-R	TGTGATGATGCTGATGCGGT
ZmPAL8-F	TCACCGTGTTCTTGACGGAG
ZmPAL8-R	TCCTCAAGCTCATGTCGTCC
ZmPAL11-F	TGGCCCTCGTGCAAAGTATC
ZmPAL11-R	TTGGTTTCCCATCTGGCGAT
ZmPAL13-F	ACGCGCCAGCATTGAGGAA
ZmPAL13-R	TGATGAACGGCACCGACAG
18S-F	CCTGCGGCTTAATTGACTC	内参基因Reference gene
18S-R	GTTAGCAGGCTGAGGTCTGG	内参基因Reference gene
ZmPAL5-WMV013-BamHI-F	AGGTCGACTCTAGAGGATCCATGGAGTGCGAGAACGGACA	构建玉米ZmPAL5过表达载体 Construction of maize ZmPAL5 overexpression vector
ZmPAL5-WMV013-SmaI-R	TCTTTGTAGTCCATCCCGGGGCCTCCACCCCCGCCACCGCAGATGGGCAGGGGCTCAC
AT3G10340-PKI1.1R-F	ATTGGACAGTTATGGAGTTACCAC	构建拟南芥CRISPR/Cas9敲除载体及菌落验证 Construction of an Arabidopsis CRISPR/Cas9 knockout vector and colony validation
AT3G10340-PKI1.1R-R	AAACGTGGTAACTCCATAACTGTC
U6.26-F	TGTCCCAGGATTAGAATGATTAGGC