苹果类受体激酶基因MdLYK1正调控对腐烂病的抗性

doi:10.16420/j.issn.0513-353x.2022-0922

摘要/Abstract

摘要：

以苹果类受体激酶基因MdLYK1（MD09G1111800）为研究对象，结合生物信息分析、功能验证和表达分析，探究其对抗腐烂病的作用和可能的作用机制。结果表明，MdLYK1与拟南芥类受体激酶基因AtLYK1同源，氨基酸序列的相似性为68.1%。将该基因在‘烟富6号’苹果果实中瞬时过表达后显著提高了果实对腐烂病的抗性。将其导入杜梨悬浮细胞，获得3个过表达细胞系。接种腐烂病菌（Valsa pyri）后，菌落在所有过表达细胞系上的生长速度显著低于野生型细胞。与野生型相比，所有过表达细胞系对Valsa pyri代谢物的敏感性显著降低。此外，MdLYK1过表达显著增强了杜梨悬浮细胞病原体相关分子模式触发的免疫反应、活性氧和茉莉酸等信号相关关键基因的上调表达。综上所述，MdLYK1正调控苹果和梨对腐烂病的抗性，且病原体相关分子模式触发的免疫反应、活性氧和茉莉酸等信号参与了其对抗性的调控过程。

关键词: 苹果, 腐烂病, 类受体激酶基因, LysM-RLK, 模式识别受体

Abstract:

The effect and potential mechanism of receptor like kinase gene MdLYK1（MD09G1111800）on the resistance to Valsa canker were investigated by bioinformatics analysis，functional verification and expression analysis. The results showed that MdLYK1 was homologous to Arabidopsis receptor kinase gene AtLYK1，with 68.1% amino acid sequence similarity. The transient expression of this gene in‘Yanfu 6’fruit significantly improved the fruit Valsa canker resistance. Three overexpressed cell lines were obtained by introducing them into the suspension cells of Pyrus betulifolia Bunge. After inoculation with Valsa pyri，the growth rate of colonies on all overexpressing cell lines was significantly lower than that of wild-type cells. All overexpression cell lines were significantly less sensitive to Valsa pyri metabolites compared to the wild type. In addition，MdLYK1 overexpression significantly enhanced the up-regulated expression of key genes related to signaling such as pathogen-associated molecular patterns triggered immunity，reactive oxygen species，jasmonic acid and other signal-related key genes were up-regulated in the suspension cells of Pyrus betulifolia Bunge. In conclusion，MdLYK1 positively regulates the resistance to apple and pear Valsa canker，and pathogen-associated molecular pattern-triggered immunity，reactive oxygen species and jasmonic acid signals are involved in the regulation of resistance.

Key words: apple, Valsa canker, receptor-like kinase gene, LysM-RLK, pattern recognition receptor

李岩妍, 郭守军, 蔡敏蕊, 孙娥, 余宏强, 蒋达吉, 左存武, 王海. 苹果类受体激酶基因MdLYK1正调控对腐烂病的抗性[J]. 园艺学报, 2023, 50(11): 2499-2508.

LI Yanyan, GUO Shoujun, CAI Minrui, SUN E, YU Hongqiang, JIANG Daji, ZUO Cunwu, WANG Hai. The Apple Receptor Like Kinase Gene MdLYK1 Positively Regulates Valsa Canker Resistance[J]. Acta Horticulturae Sinica, 2023, 50(11): 2499-2508.

图/表 6

表1 PCR和qRT-PCR引物

Table 1 Primers for PCR and quantitative real-time PCR

用途 Usage	基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
RT-PCR	MdLYK1	CTGGCGCGCCATGGGATTTCGAATCGGGTTAGGGTTCC	TCTCTAGACTGCCTTCCTGACATCAGATTGACAA
qRT-PCR	MdLYK1	TTGAGGAGGCTTTATTTTCAATTTT	GTATCACCTGAGCTCGACTTCAAA
	PbeWRKY22	CATATCCAAGGGGATATTACAGATG	GTGACTATAAAAATATTCGGGTCGG
	PbeFRK1	TCGAGTCCAAAAAACGACAGTTTAC	TTGTCAAGTTTCTGTGATGAACTCT
	PbeOXI1	CAACTTGGAAAACTCCGAGAAGTC	TTGTCAGTATCCGATAAAACGTCTC
	PbeTaNOX	TTGGAGTTGTTCGATGCGTTG	TCTTGTCCACCATGTCGAAGAAG
	PbePR1	AATCTTGTTCATTTTGGTGGGCC	AACAACCTGAGTATAATGCCCACAC
	PbePR4	GGTGAGAATTGTTGATCAGTGCAG	GTAGTTGACAATAAGGTGGCCTTG
	PbePDF1.2	GTCAGGGTTTTGTGCAAACACC	TGAAGTAGCAGAAGCAAGCAACTC
	PbeLOX1	GCTTATGTGGCTGTAAATGACTCTG	GAGGATGCAGAAGTTTGTAAATTGG

图1 苹果MdLYK1进化分析及其组织特异性表达模式 A中黑色三角表示进化树该分支所包含的基因；各分支左侧数字为自展值用来评估该分支的可信度；标尺表示序列间差异数值的单位长度。Os：水稻；At：拟南芥；Sl：番茄。

Fig. 1 Evolutionary characteristics and tissue specific expressional patterns of apple MdLYK1 The black triangle in A represents the genes contained in this branch of the evolutionary tree. The numbers on the left side of each branch are bootstrap values used to evaluate the reliability of the branch. Ruler represents the unit length of the difference value between sequences. Os：Oryza sativa；At：Arabidopsis thaliana；Sl：Solanum lycopersicum.

图2 MdLYK1正调控苹果果实对黑腐皮壳菌的抗性 *P < 0.05；** P < 0.01。下同。

Fig. 2 MdLYK1 positive regulate the resistance of apple fruits to Valsa mali *P < 0.05；** P < 0.01. The same below.

图3 MdLYK1正调控杜梨悬浮细胞对腐烂病菌Valsa pyri的抗性

Fig. 3 MdLYK1 positive regulates the resistance of Pyrus betulifolia Bunge suspension cells to Valsa pyri

图4 不同浓度黑腐皮壳菌代谢物（VpM）处理下杜梨野生型（WT）和MdLYK1过表达株系（OE11）悬浮细胞的活性

Fig. 4 Activity of suspension cells of wild-type（WT）and MdLYK1 overexpressing strains（OE11）of Pyrus betulifolia Bunge under different concentrations of Valsa pyri metabolites（VpM）treatment

图5 杜梨MdLYK1过表达细胞（OE11）和野生型（WT）在20%黑腐皮壳菌代谢物（VpM）处理后防御基因的表达

Fig. 5 Expression of defense genes in Pyrus betulifolia Bunge MdLYK1 overexpressing cells（OE11） and wild-type（WT）after 20% Valsa pyri metabolites（VpM）treatment

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