过表达ZmIBH1-1提高玉米苗期抗旱性

doi:10.3864/j.issn.0578-1752.2021.21.002

摘要/Abstract

摘要：

【目的】干旱是严重影响玉米生长发育进程的一个重要因素。挖掘玉米抗旱相关基因,通过转基因功能验证和转录组分析,解析关键基因在响应干旱胁迫过程中的分子调控机制,为抗旱分子育种和遗传改良提供理论依据。【方法】以玉米自交系B104（WT）为背景材料,利用农杆菌介导方法构建过表达ZmIBH1-1转基因株系（ZmIBH1-1-OE）;通过对转基因植株进行草铵膦抗性筛选、标记基因和目的基因PCR检测,以及运用实时荧光定量PCR检测目的基因的表达情况,鉴定阳性植株和株系;以WT和ZmIBH1-1-OE转基因株系为材料,通过干旱处理（20% PEG6000）,进行表型鉴定和耐旱生理生化指标测定,验证ZmIBH1-1的抗旱功能;通过对干旱胁迫下玉米4叶期转录组的比较分析,鉴定出差异表达的基因（differentially expressed genes,DEGs）;结合DAP-seq（DNA affinity purification sequencing）分析,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的下游靶基因,利用基因组可视化软件IGV（integrative genomics viewer）分析ZmIBH1-1蛋白结合候选靶基因的位置,然后通过Dual-Luciferase试验验证ZmIBH1-1蛋白与靶基因的调控关系。【结果】通过玉米遗传转化获得12个转化事件;T₃代中,能同时检测到标记基因Bar和目的基因ZmIBH1-1的植株有458个,实时荧光定量PCR检测结果表明,ZmIBH1-1-OE中ZmIBH1-1的表达量显著高于WT,株系3和株系8表达量最高,将其自交获得T₄代转基因株系用于后续试验。在干旱胁迫条件下,ZmIBH1-1-OE株系存活率、叶片相对含水量、叶绿素含量、可溶性蛋白含量及其生理生化指标（超氧化物歧化酶、过氧化物酶、过氧化氢酶活性）均显著高于WT,说明玉米中过量表达ZmIBH1-1赋予玉米更高的耐旱性。转录组分析结果表明,WT与ZmIBH1-1-OE株系在干旱胁迫下有1 214个差异表达基因;Gene Ontology（GO）功能富集分析结果表明,差异表达基因主要涉及生物过程、细胞组分和分子功能,如在生物过程中主要涉及到光合作用、应激响应、脱水响应等;KEGG富集分析表明,差异表达基因主要参与植物激素信号传导、新陈代谢等过程。结合转录组显著差异表达基因和DAP-Seq分析所得到ZmIBH1-1蛋白的靶基因,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的11个候选靶基因,包括2个钙信号相关基因、3个半胱氨酸代谢相关基因、1个bHLH转录因子、1个应激响应蛋白、1个谷胱甘肽转移酶、1个氧化还原过程蛋白和2个乙烯响应因子;基因组可视化结果显示ZmIBH1-1蛋白可以结合靶基因启动子区;随后通过Dual-Luciferase试验进一步表明,ZmIBH1-1蛋白可以直接作用于11个候选靶基因,其中,ZmIBH1-1蛋白可以促进ZmCa-M、ZmSYCO、ZmbHLH54、ZmGlu-r1、ZmCLPB3和ZmP450-99A2的表达,抑制ZmAGD12、ZmCYS、ZmCYSB、ZmERF-107和ZmEIN3的表达。此外,在干旱胁迫下NAC、WRKY、MYB等转录因子在ZmIBH1-1-OE和WT株系中也存在差异表达。【结论】ZmIBH1-1的过表达可以增强玉米苗期的耐旱性;ZmIBH1-1蛋白通过直接调控乙烯信号通路中的ZmERF-107和ZmEIN3的表达提高玉米的耐旱性;ZmIBH1-1蛋白通过直接调控钙信号相关基因ZmCa-M和ZmAGD12增强玉米的耐旱性;ZmIBH1-1蛋白可能通过间接调控NAC、WRKY、MYB等转录因子响应干旱胁迫。

关键词: 玉米, 干旱胁迫, ZmIBH1-1, RNA-Seq, 转录因子, 基因表达

Abstract:

【Objective】Drought is an important factor that affects the growth and development of maize seriously. Through the mining of genes related to drought resistance in maize, transgene function verification and transcriptome analysis, analyzing the molecular regulation mechanism of key genes in response to drought stress, this paper provides the theoretical basis for drought resistance molecular breeding and genetic improvement. 【Method】In this study, the maize inbred line B104 (wild-type, WT) was used as the background to construct the ZmIBH1-1 overexpression (ZmIBH1-1-OE) transgenic line by Agrobacterium-mediated method. The transgenic plants and lines were identified via screening transgenic plants for glufosinate-ammonium resistance, PCR detection of marker gene and target gene and expression analysis of target gene by qRT-PCR. We used the ZmIBH1-1-OE and WT transgenic lines as materials. Through drought treatment (20% PEG6000), phenotype identification and drought tolerance physiological and biochemical index determination were carried out to verify the drought resistance function of ZmIBH1-1; RNA-Seq was used to identify differentially expressed genes (DEGs) under drought stress at the 4-leaf stage; Combined with DAP-seq (DNA affinity purification sequencing) analysis, it is preliminarily determined that ZmIBH1-1 protein directly regulates downstream target genes related to drought resistance, and IGV (Integrative Genomics Viewer) was used to analyze the position of the ZmIBH1-1 protein binding candidate target gene, and then the Dual-Luciferase assay was used to verify the regulatory relationship between ZmIBH1-1 protein and target genes. 【Result】12 transformation events were obtained by genetic transformation of maize. In the T₃ generation, there were 458 plants in which the marker gene Bar and the target gene ZmIBH1-1 were simultaneously detected. The results of qRT-PCR showed that the expression level of ZmIBH1-1 in ZmIBH1-1-OE lines was significantly higher than that of WT and the expression levels of transformation events 3 and 8 were the highest, which were self-crossed to obtain T₄ generation for subsequent experiments. Under drought stress, the survival rate, the relative water content, the chlorophyll content, soluble protein content and the physiological and biochemical indicators (superoxide dismutase, peroxidase, catalase activity) of ZmIBH1-1-OE were higher than those of WT significantly, which indicating that the overexpression of ZmIBH1-1 in maize confers higher drought tolerance. The RNA-Seq results showed that there were 1 214 DEGs between WT and ZmIBH1-1-OE lines. Gene Ontology (GO) analysis showed that DEGs were mainly involved in biological processes, cell components and molecular functions, such as photosynthesis, stress response, dehydration response, etc. in biological processes; KEGG enrichment analysis showed that DEGs were mainly involved in the signal transduction of plant hormones, the metabolism and other processes. Combining the significantly DEGs of RNA-Seq and the target genes of ZmIBH1-1 obtained from DAP-seq analysis, it is preliminarily identified 11 candidate target genes related to drought resistance that may be directly regulated by ZmIBH1-1, including 2 calcium signal related genes, 3 cysteine metabolism related genes, 1 bHLH transcription factor, 1 stress response protein, 1 glutathione transferase, 1 redox process protein and 2 ethylene response factor; Integrative genomics viewer showed that ZmIBH1-1 protein could bind to the promoters of the target genes; Subsequent Dual-Luciferase assay further showed that ZmIBH1-1 protein can directly act on 11 candidate target genes, of which, ZmIBH1-1 directly binds to the promoters of ZmCa-M, ZmSYCO, ZmbHLH54, ZmGlu-r1, ZmCLPB3 and ZmP450-99A2 to promote their expression, and directly binds to the promoters of ZmAGD12, ZmCYS, ZmCYSB, ZmERF-107 and ZmEIN3 to repress their expression. In addition, transcription factors such as NAC, WRKY and MYB also differentially expressed between WT and ZmIBH1-1-OE under drought stress. 【Conclusion】The overexpression of ZmIBH1-1 can enhance the drought tolerance of maize; ZmIBH1-1 improves the drought tolerance of maize by directly regulating the expression of genes ZmERF-107 and ZmEIN3 in the ethylene signaling pathway; ZmIBH1-1 enhances the drought tolerance of maize by directly regulating the calcium signal-related genes ZmCa-M and ZmAGD12; ZmIBH1-1 may indirectly regulate NAC, WRKY, MYB and other transcription factors in response to drought stress.

Key words: maize, drought stress, ZmIBH1-1, RNA-Seq, transcription factor, gene expression

朱芳芳,董亚辉,任真真,王志勇,苏慧慧,库丽霞,陈彦惠. 过表达ZmIBH1-1提高玉米苗期抗旱性[J]. 中国农业科学, 2021, 54(21): 4500-4513.

ZHU FangFang,DONG YaHui,REN ZhenZhen,WANG ZhiYong,SU HuiHui,KU LiXia,CHEN YanHui. Over-expression of ZmIBH1-1 to Improve Drought Resistance in Maize Seedlings[J]. Scientia Agricultura Sinica, 2021, 54(21): 4500-4513.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)
Bar-F	CATCGAGACAAGCACGGTC
Bar-R	AAACCCACGTCATGCCAGTT
ZmIBH-F	CAATTACATTTACAATTACCATGGTCATGGCCAGGAAGAGGAC
ZmIBH-R	CTCTCTAGACTCACCTAGGATCCTCATTGGGCGGAGAAG
qIBH-F	AGGAACCACCGCCAAACC
qIBH-R	GCHTCTCCGCAGCAGGAC
Tublin-F	CCGCTATCTCCGTCGC
Tublin-R	GTTCTTGGATGGCGGTCG

样品 Sample	正常条件Normal					干旱胁迫Drought stress
样品 Sample	原始reads Raw reads	过滤后的reads Clean reads	过滤后的 reads所占比 Reads keep rate (%)	比对上的 reads Mapped reads	比对上的 reads占比 Mapped reads rate (%)	原始reads Raw reads	过滤后的 reads Clean reads	过滤后的 reads所占比 Reads keep rate (%)	比对上的reads Mapped reads	比对上的 reads占比 Mapped reads rate (%)
WT-1	5407756	5032404	93.05	4492503	89.27	3870308	3532833	91.28	3128759	88.56
WT-2	7232533	6586333	91.06	5987305	90.90	4119004	3813323	92.57	3482561	91.32
OE-1	8523635	7780152	91.27	6893250	88.60	6322179	5776743	91.37	5367210	92.91
OE-2	7647821	6977539	91.23	6289035	90.13	6994016	6490634	92.80	5901352	90.92