甘蔗抗褐锈病Bru1区域的鉴定及候选抗病基因的功能分析

Identification of the Bru1 Genomic Region for Brown Rust Resistance and Functional Analysis of Candidate Resistance Genes in Sugarcane

【目的】甘蔗褐锈病是由黑顶柄锈菌(Puccinia melanocephala H. Sydow&P. Sydow)引起的最具破坏性的真菌病害之一,能造成蔗糖分降低10%—36%,严重威胁甘蔗产业发展。已有研究揭示抗褐锈病主效基因定位于Bru1区域,克隆其关键候选基因,并进行功能研究,为选育抗褐锈病甘蔗新品种提供重要的候选基因资源。【方法】利用PacBio SequelⅡ测序平台获得甘蔗栽培品种ROC22的contig水平基因组信息,鉴定到抗褐锈病Bru1区域,对其进行基因注释和克隆,分析其组织特异性和抗、感褐锈病甘蔗品种中的表达模式及其在烟草叶片的亚细胞定位和瞬时表达。【结果】基于Bru1位点区域紧密连锁的标记R12H16和9O20-F4,从该区域中注释到33个基因,结合典型抗病基因结构域,共筛选获得5个候选抗病基因,分别为Brrg99、Brrg103、Brrg108、Brrg115和Brrg116。以甘蔗栽培品种ROC22的cDNA为模板,克隆获得Brrg116的全长序列729 bp,编码242个氨基酸残基。将该基因序列分别比对甘蔗热带种和割手密种及二倍体近缘种高粱的基因组数据库,发现其与热带种和割手密种的同源基因序列相似性很高,达到98%以上,而与高粱的相似性为93.77%。系统进化树揭示该基因起源甘蔗割手密种。实时荧光定量PCR检测表明,Brrg116在甘蔗栽培品种的多个组织中组成型表达,尤其是在+1叶中的表达量最高,是蔗芽的9.8倍;在褐锈病菌侵染抗、感甘蔗栽培品种的6和72 h时,呈现显著差异表达。亚细胞定位结果显示,该基因编码蛋白定位于细胞膜上。在本氏烟(Nicotiana benthamiana)叶片中瞬时过表达Brrg116,其后接种茄病镰刀菌蓝色变种,二氨基联苯胺法(3,3′-diaminobenzidine,DAB)染色逐渐加深,超敏反应相关基因、水杨酸信号和乙烯信号途径中的相关基因持续上调表达。【结论】Brrg116在甘蔗不同组织中组成型表达,且在受褐锈病菌侵染的抗病甘蔗栽培品种中呈现快速的诱导表达。此外,过表达Brrg116引发水杨酸和乙烯等激素信号通路产生防御响应,推测该基因可能在提高植物的抗病性方面发挥重要调控作用。

【Objective】Sugarcane brown rust,caused by Puccinia melanocephala H.Sydow&P.Sydow,is one of the most destructive fungal diseases in sugarcane industry,leading to a reduction in sucrose content by 10%to 36%.Previous studies revealed that the major gene(s)for brown rust resistance was located in the Bru1 genomic region.The cloning of crucial candidate genes and their functional investigation should provide important candidate gene resources for breeding new sugarcane cultivars resistant to brown rust.【Method】In this study,the contig-level genomic information of sugarcane cultivar ROC22 was obtained by utilizing the PacBio Sequel sequencing platform,and the Bru1 region associated with brown rust candidate resistance gene was identified,Ⅱannotated,cloned,and analyzed for tissue specificity,expression patterns in resistant and susceptible sugarcane cultivars,subcellular localization,and transient overexpression.【Result】The results demonstrated that,firstly,using tightly associated molecular markers R12H16 and 9O20-F4 within the Bru1 region,a total of 33 genes were annotated from this region,and five candidate resistance genes(Brrg99,Brrg103,Brrg108,Brrg115,and Brrg116)were selected based on the typical/conserved domains of the resistance genes.Secondly,the full-length sequence cDNA sequence of the Brrg116 gene with an open reading frame of 729 bp and encoding 242 amino acid residues,was cloned from sugarcane cultivar ROC22.The gene sequence was aligned with the genomic databases of Saccharum spontaneum,S.officinarum,and the closely related diploid species sorghum.A high degree of sequence similarity was observed between S.spontaneum and S.officinarum,exceeding 98%.In contrast,its similarity with sorghum was 93.77%.Phylogenetic tree analysis suggests that this gene may originate from S.spontaneum species during sugarcane domestication.qRT-PCR analysis showed its constitutive expression in various tissues of sugarcane cultivars,particularly with the highest expression level in the+1 leaf,which was 5.2 times higher than in the bud.Furthermore,significantly differential expression of Brrg116 was observed at 6 h and 72 h post-inoculation with the brown rust pathogen in resistant and susceptible sugarcane cultivars.Subcellular localization analysis indicated that the encoded protein of this gene was located on the cell membrane.Finally,the Brrg116 gene was transiently overexpressed in Nicotiana benthamiana leaves,followed by inoculation with Fusarium solani var.coeruleum.The color of Nicotiana benthamiana leaves showed a gradual deepening phenotype by 3,3'-diaminobenzidine(DAB)staining.The genes related to hypersensitive response,salicylic acid,and ethylene synthesis had a sustained upregulation pattern,evidencing that the expression of these genes can enhance plant disease resistance.【Conclusion】Brrg116 was constitutively expressed in different sugarcane tissues and in response to brown rust pathogen infection,it showed a rapidly induced expression pattern in the resistant sugarcane cultivar.Overexpression of Brrg116 could trigger defense responses through hormone signaling pathways such as salicylic acid and ethylene.It is thus hypothesized that this gene may play a significant regulatory role in enhancing plant disease resistance.