以红肉苹果‘紫红3号’愈伤组织为试材,克隆了细菌鞭毛蛋白受体基因MdFSL2,研究了其与拟南芥AtFLS2对细菌鞭毛蛋白敏感性差异以及对苹果轮纹病抗性的影响。进化树分析表明,MdFLS2与拟南芥AtFLS2亲缘关系较远,处于不同的进化树分支,与梨PbFLS2的亲缘关系最近。时空表达特异性研究表明,在苹果叶片中MdFLS2表达不随组织衰老而产生差异,能够被外源SA处理诱导,不受IAA、ACC处理影响;在根系中表达量最高,而在花与果实中表达量较低。在拟南芥中异源过表达MdFLS2,显著抑制植株生长,并出现叶片边缘枯死或细胞死亡的表型。在转基因拟南芥中受SA诱导的病程相关基因AtPR1、AtPR2和AtPR5,及衰老相关基因AtORE1和AtNAP的表达水平均显著高于野生型。为研究MdFLS2是否具有感知细菌鞭毛蛋白的能力,进行拟南芥根系生长抑制试验,MdFLS2超表达恢复了细菌鞭毛蛋白N端22个保守氨基酸肽段（flg22）对拟南芥fls2突变体根系生长的抑制作用,但flg22分别处理30和60 min,或20和40 min,flg22诱导的标志性基因表达水平及MAPK激酶活化水平在过表达MdFLS2的fls2突变体中显著低于同样处理的野生型。超表达MdFLS2提高了拟南芥叶片对轮纹病菌的抗性,并增强了拟南芥突变体fls2对假单胞菌DC3000的抗性。研究结果说明苹果MdFLS2是一个有功能的免疫相关基因,MdFLS2与AtFLS2在具体执行功能与作用机制上可能存在差异。
MdFLS2（MDP0000254122 as registered in GDR）encoding a putative receptor perceiving bacterial flagellin was cloned from the apple callus of‘Zihong 3＇. Phylogeny analysis showed that MdFLS2 bears distantly related homologies with FLS2 in Arabidopsis thaliana（AtFLS2,At5g46330.1）, but showed the closest homologies with FLS2 in Pyrus bretshneideri. Temporal and spatial expression analysis indicated that there was no observed difference on the expressional level of this gene in leaves with different senescence degree. But its expression could be significantly up-regulated by exogenously provided salicylic acid（SA）but not IAA and ACC. The gene was expressed predominantly in root with subsequently sequential declining in stem,leaf,and flower and fruit. When heterologously overexpressed in Arabidopsis,MdFLS2 significantly retarded the seedling growth and caused the death and wilt of certain leaves. The expression of pathogenesis related genes AtPR1,AtPR2 and AtPR5 which were regulated by SA are up-regulated similarly as those senescence regulated genes AtORE1 and At NAP. Overexpression of MdFLS2 in the Arabidopsis fls2 mutant restored the inhibition of root elongation by flg22,while the induction of flg22-induced marker genes 30 and 60 minutes after flg22 treatment and the initiation of MAPK signaling pathway 20 and 40 minutes after flg22 treatment in MdFLS2 overexpression transgenic plants was significantly lower than that of wild type plants. Moreover, the disease resistance of Arabidopsis leaves against the apple ring rot causal fungi Botryosphaeria dothidea was significantly enhanced by MdFLS2 overexpression and the immune responses were also significantly strengthened compared with the fls2 mutant during the infection of Arabidopsis leaves with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our result suggested that MdFLS2 functions as an immune gene but displayed differences in the specific implementation of the function and mechanism of action.
Acta Horticulturae Sinica
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