三明野生蕉β-1,3葡聚糖酶基因克隆及其低温下SA处理后的表达分析

Cloning and Expression Analysis under Cold and SA Treatments of β-1,3-glucanase Genes from the Wild Banana in Sanming City

以三明野生蕉（Musaspp.AB group）为材料,利用RT-PCR和RACE技术,克隆获得β-1,3葡聚糖酶5个基因（Mugsp1.2~Mugsp5）的cDNA和DNA序列。序列和生物信息学分析表明Mugsp1.2、Mugsp2、Mugsp3、Mugsp4和Mugsp5的ORF长度分别为1 020、1 047、999、1 023和960bp,分别编码339、348、332、340和319个氨基酸;Mugsp1.2、Mugsp2和Mugsp4蛋白具有N端和C端（CTPP）信号肽结构,推测为Ⅰ类β-1,3葡聚糖酶,而Mugsp3只含有N端信号肽,无CTPP结构,Mugsp5则不具有信号肽结构;构建Mugsp1.2、Mugsp2、Mugsp4瞬时表达载体并转化洋葱表皮的亚细胞定位观察结果显示,常温下Mugsp1.2、Mugsp2和Mugsp4主要在细胞膜、细胞质上表达,而经过8℃低温处理后Mugsp1.2、Mugsp2和Mugsp4均能够转移至细胞核表达;β-1,3葡聚糖酶基因在不同低温处理下的实时荧光定量（qRT-PCR）检测结果显示,β-1,3葡聚糖酶基因均能响应低温胁迫,是低温胁迫相关基因,但基因间表达水平存在差异;低温下SA处理后的定量结果显示,SA处理会推迟β-1,3葡聚糖酶基因的表达。

The cDNA and DNA sequences of β-1,3-glucanase genes（Mugsp1.2-Mugsp5）were cloned from the leaves of the wild banana（Musaspp.）in Sanming City using RT-PCR and RACE methods.Sequences and bioinformatics analysis showed that the ORF sequences of Mugsp1.2,Mugsp2,Mugsp3,Mugsp4 and Mugsp5were 1 020,1 047,999,1 023 and 960bp,respectively,which encoded 339,348,332,340 and 319amino acids,respectively.Mugsp1.2,Mugsp2 and Mugsp4contained both N-terminal and C-terminal（CTPP）signal peptide,suggesting they belong toⅠclass ofβ-1,3-glucanase,while Mugsp3 contained N-terminal without CTPP signal peptide,and Mugsp5 contained neither N-terminal nor CTPP signal peptide.We constructed the expression vectors of Mugsp1.2,Mugsp2,Mugsp4 and expressed the genes in onion epidermal cells.The results of subcellular localization showed that Mugsp1.2,Mugsp2,Mugsp4 were expressed on plasma membranes and cytoplasm at room temperature,respectively,while all Mugsp1.2,Mugsp2 and Mugsp4could transfer to cell nucleus under 8 ℃.The expressions ofβ-1,3-glucanase genes underdiffierent tempertaures were dectected by qRT-PCR.The results revealed that allβ-1,3-glucanase genes responded to cold stress,but their expression levels were differed from each other.SA treatment under low temperature would delayed the expression ofβ-1,3-glucanase genes.