草莓（Fragaria spp.）是蔷薇科（Rosaceae）小型浆果植物，果实香甜，营养丰富，深受人们的喜爱。灰霉病（Botrytis cinerea）是影响草莓生长发育的主要病害之一，能够感染草莓植株的所有组织，最终导致植株的死亡。草莓感染灰霉病后不仅品质会受到严重的影响，而且产量会减少10%~50%，在生产上带来巨大的经济损失。NBS-LRR（Nucleotide binding site-leucine rich repeats）基因是植物体内最重要的一类抗病基因，介导植物产生防御反应，在应对自然界的病虫害侵扰中发挥着重要作用。森林草莓（F. vesca）是草莓属中的二倍体野生种，分布范围较广，遗传变异性较高。根据已构建的森林草莓NBS-LRR基因家族系统进化树，发现3个抗病候选基因FvNLR1、FvNLR2、FvNLR3均分布于较短的进化枝上，推测它们属于快速进化模式。本文主要研究这3个抗病候选基因的结构特点以及在欧洲不同生态型森林草莓中发生的遗传变异，其次对抗灰霉病候选基因FvNLR2进行了功能验证，并且分析了转基因植物中病程相关基因的表达模式以及过表达FvNLR2基因后对其产生的影响，研究结果如下：

1. 分别比较了FvNLR1、​​FvNLR2、FvNLR3基因在不同欧洲森林草莓生态型中的基因及其编码的蛋白质序列差异，结果显示FvNLR3基因的遗传变异程度最大，FvNLR1基因的遗传变异程度最小，且3个基因的遗传变异部分发生在其编码的结构域中。系统进化树显示取样自同一国家的生态型，其NBS-LRR基因更偏向于聚类在同一个进化枝上，说明它们之间核苷酸差异度较小。分析这3个基因所受到的选择压力，发现FvNLR1和FvNLR3基因及大部分结构域的平均Ka/Ks均小于1，揭示了纯化选择在基因进化过程中的重要作用。3个NBS-LRR基因中内含子数量不同且数量均偏少。在蛋白保守基序分析中，3个NBS-LRR基因中均预测出了NBS结构域保守基序激酶1（P-loop）、激酶-2、GLPL以及LRR基序。在3个NBS-LRR基因启动子中共预测到了32个顺式作用元件，包括大量防御和应激等作用元件，推测这3个NBS-LRR基因在植物抗病过程中发挥重要作用。

2. 在灰霉菌侵染草莓后96h发现，FvNLR2基因表达量显著高于FvNLR1和FvNLR3基因，提示FvNLR2基因具有潜在抗灰霉病功能。亚细胞定位结果显示FvNLR2基因定位在细胞膜，这与RPW8-NBS-LRR基因编码跨膜转运蛋白，具有传递抗病信号等功能相印证。通过农杆菌介导的瞬时转化，成功在森林草莓和凤梨草莓中对FvNLR2基因进行了过表达和沉默，接种灰霉菌5天后观察病斑大小均发现：过表达FvNLR2基因植物材料中病斑小于对照，而沉默FvNLR2基因植物材料中病斑大于对照，说明FvNLR2基因能够正调控草莓对灰霉病的抗性。通过叶盘法在烟草中进行稳定遗传转化，成功鉴定出8株FvNLR2基因过表达转基因烟草，接种灰霉菌5天后，过表达植株叶片病斑显著小于对照植株叶片，这与在草莓中瞬时转化的结果一致，再次验证FvNLR2基因具有抗灰霉病功能。对接种灰霉菌24 h后的转基因烟草叶片进行DAB（3,3N-Diaminobenzidine Tertrahydrochloride）染色后发现转基因烟草叶片中积累了大量的活性氧。

3. 植物病程相关蛋白（Pathogenesis-related Protein，PRs）在植物抵御病原菌侵染以及应对非生物胁迫时发挥着重要作用，且PR基因会响应抗病基因的过表达，提高植物抵抗病原菌侵染的能力。研究转基因草莓和烟草中PR基因的表达水平，结果显示FvNLR2抗病基因在植物体内过表达后能够促进大部分PR基因的上调。在对照植物中PR1、PRH1和PR4基因表现了出对灰霉菌的强烈响应，在转基因植株中，FvNLR2基因的过表达加强了这种响应，推测FvNLR2基因可能通过调节抗病相关蛋白的表达水平从而使植物产生防御反应。

Strawberry (Fragaria spp.) is a small berry plant of the Rosaceae, which is sweet, nutritious and loved by people. Gray mold (Botrytis cinerea), one of the main diseases affecting the growth and development of strawberries, can infect all tissue parts of strawberry, and eventually lead to the death. After strawberries are infected with B. cinerea, not only the quality will be seriously affected, but also the yield will be reduced by 10%~50%, which will bring huge economic losses in production. NBS-LRR (Nucleotide binding site-leucine rich repeats) gene is one of the most important resistance genes in plants, which mediates the disease resistance response of plants and plays an important role in coping with pest and disease infestation in nature. Woodland strawberry (F. vesca) is a diploid wild species in the genus, which is widely distributed, high genetic variability. According to the phylogenetic tree of the NBS-LRR gene family of woodland strawberry, the three disease resistance candidate genes FvNLR1, FvNLR2 and FvNLR3 were distributed on shorter clades, and it is speculated that they are in a mode of rapid evolution. In this paper, the structural characteristics of these three disease resistance candidate genes and the genetic variation in different ecological woodland strawberries in Europe were studied. Secondly, the function of the candidate gene FvNLR2 on gray mold was verified. Finally, the expression pattern of disease-related genes in transgenic plants and the effects of overexpression of FvNLR2 genes were analyzed, and the results were as follows:

1. The gene and the protein sequence of FvNLR1, FvNLR2 and FvNLR3 genes in different European woodland strawberry ecotypes were compared, and the results showed that the genetic variation of FvNLR3 gene was the largest, the genetic variation of FvNLR1 gene was the smallest, and the genetic variation of the three genes occurred in the domain they encoded. The phylogenetic tree shows that NBS-LRR genes sampled from the same country are more inclined to cluster on the same clade, indicating that there is less nucleotide difference between them. The selection pressure of these three genes was analyzed, and it was found that the average Ka/Ks of FvNLR1 and FvNLR3 genes and most domains were less than 1, revealing the important role of purification selection in gene evolution. The number of introns in the three NBS-LRR genes was different and the number was small. In the protein conserved motif analysis, NBS domain conserved motif kinase 1 (P-loop), kinase-2, GLPL and LRR motifs were predicted in all three NBS-LRR genes. A total of 32 cis-acting elements were predicted in the promoters of the three NBS-LRR genes, including a large number of action elements such as defense and stress, and it was speculated that these three NBS-LRR genes played an important role in the process of plant disease resistance.

2. 96 h after B. cinerea infestation F. vesca, it was found that the expression of FvNLR2 gene was significantly higher than that of FvNLR1 and FvNLR3 genes, indicating that FvNLR2 gene had potential function that resistance to gray mold. The results of subcellular localization showed that the FvNLR2 gene was localized in the cell membrane, which was confirmed by the RPW8-NBS-LRR gene encoding a transmembrane transporter and transmitting disease resistance signals. Through the transient transformation mediated by arobacterium, FvNLR2 gene was successfully overexpressed and silenced in F. vesca and F. ananassa, and the size of disease spots was observed after 5 days of inoculation with B. cinerea and found that the results were similar: the disease spots in the overexpressed FvNLR2 gene plant material were smaller than the control, while the disease spots in the silenced FvNLR2 gene plant material were larger than the control, indicating that the FvNLR2 gene could positively regulate the resistance to gray mold. Eight overexpression transgenic tobacco strains of FvNLR2 gene were successfully identified by stable genetic transformation in tobacco by leaf method, and the leaf disease spots of overexpressed plants were significantly smaller than those of control plants after 5 days of inoculation with B. cinerea. This is consistent with the results of transient transformation in strawberries, and once again verifies that the FvNLR2 gene has function that resistance to gray mold. DAB (3,3N-Diaminobenzidine Tertrahydrochloride) staining of transgenic tobacco leaves after 24 h inoculation with B. cinerea found that a large amount of reactive oxygen species accumulates in transgenic tobacco leaves.

3. Plant pathogenesis-related proteins (PRs) play an important role in plants resisting pathogenic bacteria infection and abiotic stress, moreover, PR genes will respond to the overexpression of disease resistance genes and improve the ability of plants to resist pathogenic bacteria The expression levels of PR genes in transgenic strawberries and tobacco were studied, and it was found that FvNLR2 disease resistance genes could promote the upregulation of most PR genes after overexpression in plants. The PR1、PRH1and PR4 genes showed a strong response to B. cinerea in control plants, and in transgenic plants, overexpression of the FvNLR2 gene enhanced this response, and it was speculated that the FvNLR2 gene may cause a defensive response in plants by regulating the expression level of disease-related proteins.

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