苹果MdMYB116对白粉病的抗性研究

doi:10.16420/j.issn.0513-353x.2021-0060

摘要/Abstract

摘要：

以抗白粉病的‘嘎拉’苹果为试材,克隆得到MdMYB116的cDNA全长序列。该基因的开放阅读框为900 bp,编码299个氨基酸,含有2个保守结构域。系统进化树结果表明,MdMYB116与梨PbMYB108-like的相似性最高,为93.38%。亚细胞定位试验显示MdMYB116定位在细胞核内。MdMYB116接种苹果白粉病菌6 ~ 12 h表达量上升,并达到高峰。酵母转录激活验证得出MdMYB116全长含有自激活活性。将MdMYB116异源转化拟南芥,得到的转基因植株接种白粉病菌处理后,其抗性显著高于野生型和pad4突变体植株。组织化学染色观察发现,与野生型和pad4突变体植株相比,转基因株系活性氧积累水平更高,死细胞数量和胼胝质积累也更多,说明转基因株系存在较严重的过敏致死情况,证明MdMYB116参与植株对白粉病的免疫调节反应。通过分析相关抗病基因的表达结果,说明MdMYB116通过参与SA和/或MeJA的信号途径增强防御反应。

关键词: 苹果, MdMYB116, 白粉病, 功能分析

Abstract:

The full-length cDNA sequence of MdMYB116 was cloned from the powdery mildew resistant‘Gala’apple. The open reading frame of this gene was 900 bp,encoded 299 amino acids and contained two conserved domains. The phylogenetic tree results showed that MdMYB116 shared close relationship with PbMYB108-like in pear,with a identity of 93.38%. Subcellular localization assay evidenced that MdMYB116 was targeted to the nucleus. The expression level of MdMYB116 increased gradually from 6 h to 12 h and it was induced to a peak at 12 h after inoculation with powdery mildew. Yeast transcriptional activation verification revealed that the full length of MdMYB116 exhibited self-activating activity. MdMYB116 was transformed into Arabidopsis thaliana heterologously,and transgenic plant was obtained. After being inoculated with powdery mildew,it was found that the resistance of the transgenic lines was significantly higher than that of the wild type and pad4 mutant plants. Histochemical staining assays displayed that compared with wild type and pad4 mutant plants,the transgenic lines had a higher level of reactive oxygen species（ROS）burst,and more dead cells and calluses accumulation,indicating that the transgenic lines had a serious allergic lethal situation,which proved that MdMYB116 participated in the plant^’s powdery mildew immunomodulatory response. Expression analysis of some disease related genes speculated that MdMYB116 may enhanced defense responses though SA and/or MeJA signal pathways.

Key words: apple, MdMYB116, powdery mildew, functional analysis

中图分类号:

S661.1

吉苗苗, 万叶, 张一平, 马海, 王西平, 高华. 苹果MdMYB116对白粉病的抗性研究[J]. 园艺学报, 2021, 48(11): 2133-2145.

JI Miaomiao, WAN Ye, ZHANG Yiping, MA Hai, WANG Xiping, GAO Hua. Studies on the Resistance of Apple MdMYB116 to Powdery Mildew[J]. Acta Horticulturae Sinica, 2021, 48(11): 2133-2145.

图/表 11

表1 本研究中所用的引物

Table 1 Primers used in this study

引物用途	引物名称	序列（5′-3′）
Useage	Primer name	Sequence
基因克隆 Gene cloning	MdMYB116-Clone	F:ATGTCGACTAAGACTAAAACCCTAA
基因克隆 Gene cloning	MdMYB116-Clone	R:CTACATTTCGTCCGTGTTCCATATG
过表达/亚细胞定位 Overexpression/ subcellular localization	MdMYB116-OE	F:GGTACCATGTCGACTAAGACTAAAACCCTAA
过表达/亚细胞定位 Overexpression/ subcellular localization	MdMYB116-OE	R:GGATCCCATTTCGTCCGTGTTCCATATG
酵母双杂交 Yeast two-hybrid	MdMYB116-BD	F:ATGGCCATGGAGGCCGAATTCATGTCGACTAAGACTAAAACCCTAA
	MdMYB116-BD	R:CCGCTGCAGGTCGACGGATCCCTACATTTCGTCCGTGTTCCATATG
	MdMYB116-D1-BD	F:ATGGCCATGGAGGCCGAATTCTGGACTCTTGAAGAAGACTCTCTTC
	MdMYB116-D1-BD	R:CCGCTGCAGGTCGACGGATCCCATTTCGTCCGTGTTCCATATGCTA
	MdMYB116-D2-BD	F:ATGGCCATGGAGGCCGAATTCTGGTCGAAAATCGCGCAATA
	MdMYB116-D2-BD	R:CCGCTGCAGGTCGACGGATCCCATTTCGTCCGTGTTCCATATGCTA
	MdMYB116-D3-BD	F:ATGGCCATGGAGGCCGAATTCTGGATGCCAAGGTTGCAGCA
	MdMYB116-D3-BD	R:CCGCTGCAGGTCGACGGATCCCATTTCGTCCGTGTTCCATATGCTA
	MdMYB116-D4-BD	F:ATGGCCATGGAGGCCGAATTCATGTCGACTAAGACTAAAACCCTAA
	MdMYB116-D4-BD	R:CCGCTGCAGGTCGACGGATCCCGATATTGTGGCTGTAAAATGTGGG
	MdMYB116-D5-BD	F:ATGGCCATGGAGGCCGAATTCATGTCGACTAAGACTAAAACCCTAA
	MdMYB116-D5-BD	R:CCGCTGCAGGTCGACGGATCCTTTCTGCACCCGTGTTCTCCAGTAG
实时荧光定量分析 qRT-PCR	MdMYB116-RT	F:TGATAAGGTGCTATTGGATGC
	MdMYB116-RT	R:TGTAAAATGTGGGAAGGTGTT
	AtICS1	F:CTTCCGTGACCTTGATCCTTTCT
	AtICS1	R:CAGCGATCTTGCCATTAGGATC
	AtPR1	F:AACTACGCTGCGAACACGTG
	AtPR1	R:TCACTTTGGCACATCCGAGTC
	AtLOX3	F:TCTCCGTACAACAAGCGTTGG
	AtLOX3	R:GCGTCCGTCTAGCGCATTAAT
	AtMYC2	F:TCATACGACGGTTGCCAGAA
	AtMYC2	R:AGCAACGTTTACAAGCTTTGATTG
内参基因 Reference gene	AtActin1	F:TCAATCCAGGAGATGTTTAGG
	AtActin1	R:ACTGCTGGTACTCTGCGACA
	MdTubulin	F:AGGATGCTACAGCCGATGAG
	MdTubulin	R:GCCGAAGAACTGACGAGAATC

图1 苹果MdMYB116的克隆

Fig. 1 Gene cloning of MdMYB116

图2 MdMYB116与其他物种的氨基酸序列对比

Fig. 2 Comparison of amino acid sequences of MdMYB116 with other species

图3 MdMYB116和其他物种的MYB亲缘关系分析

Fig. 3 Phylogenetic analysis of MdMYB116 and from MYB with other species

图4 ‘嘎拉’苹果接种白粉病菌后MdMYB116的表达分析

Fig. 4 MdMYB116 expression pattern in‘Gala’apple after powdery mildew inoculation

图5 MdMYB116在烟草表皮细胞的定位情况

Fig. 5 MdMYB116 was localized in the tobacco leaf epidermal cells

图6 MdMYB116基因的转录活性分析

Fig. 6 Analysis of autoactivation of MdMYB116

图7 转基因株系（L1 ~ L3）、突变体（pad4）及野生型（WT）接种白粉病菌后的表型

Fig. 7 Phenotypes of transgenic lines（L1-L3）,mutants（pad4）and wild type（WT）after powdery mildew inoculation

图8 转基因株系（L1 ~ L3）、突变体（pad4）及野生型（WT）的DAB、NBT染色情况

Fig. 8 Histochemical staining of DAB and NBT of transgenic lines（L1-L3）,mutants（pad4）and wild type（WT）

图9 转基因株系（L1 ~ L3）、突变体（pad4）及野生型（WT）的台盼蓝、苯胺蓝染色情况

Fig. 9 Histochemical staining of trypan blue and aniline blue of transgenic lines（L1-L3）,mutants（pad4）and wild type（WT）

图10 MdMYB116转基因拟南芥株系（L1 ~ L3）和野生型（WT）接种白粉病菌后防御基因的表达量变化

Fig. 10 Analysis of quantitative expression of defense genes in MdMYB116 transgenic Arabidopsis thaliana lines（L1-L3）and wild type（WT）after powdery mildew inoculation

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