【摘要】：晚期胚胎富集蛋白(late embryogenesis abundant proteins,LEA)是一種親水、熱穩(wěn)定性蛋白,與植物抗逆性密切相關(guān),研究LEA家族基因?qū)﹁F皮石斛抗逆性研究具有重要意義。本研究以鐵皮石斛(Dendrobium officinale)種子為材料,采用RACE技術(shù)獲得全長cDNA,命名為DoLEA2(Gen Bank登錄號(hào):KY626329)。該基因全長cDNA為1 224 bp,編碼313個(gè)氨基酸,具有晚期胚胎富集蛋白(LEA_2)結(jié)構(gòu)域和水分脅迫高敏感響應(yīng)結(jié)構(gòu)域,所編碼蛋白與其他物種LEA2蛋白具有較高相似性,在進(jìn)化上與單子葉植物聚為一支,且與蝴蝶蘭(Phalaenopsis aphrodite)親緣關(guān)系最近。鐵皮石斛DoLEA2在葉的表達(dá)量最高,根和莖次之。該基因在Escherichia coli BL21(DE3)中能夠高效表達(dá),其優(yōu)化表達(dá)條件為:37℃添加終濃度0.5 mmol·L~(-1)的異丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)誘導(dǎo)4 h。鹽脅迫條件下與對照組相比,重組pET28a-DoLEA2菌株表現(xiàn)出較好的耐鹽性。本研究首次獲得了鐵皮石斛LEA2全長cDNA序列,驗(yàn)證了其耐鹽脅迫功能,為進(jìn)一步研究鐵皮石斛抗逆性分子機(jī)制提供科學(xué)依據(jù)。
[Abstract]:Late embryo enriched protein (late embryogenesis abundant proteins,LEA) is a hydrophilic and thermostable protein, which is closely related to plant stress resistance. It is of great significance to study the stress resistance of Dendrobium candidum (Dendrobium candidum) by studying LEA family genes. In this study, (Dendrobium officinale) seeds of Dendrobium candidum were used as materials and RACE technique was used to obtain the full length cDNA, named DoLEA2 (Gen Bank accession number: KY626329). The full-length cDNA of this gene is 1 224 bp, encoding 313 amino acids, with late embryo enriched protein (LEA_2) domain and water stress sensitive domain. The encoded protein is similar to LEA2 protein of other species. The phylogenetic relationship with monocotyledonous plants was similar to that of Phalaenopsis (Phalaenopsis aphrodite). The DoLEA2 expression of Dendrobium candidum was the highest in leaves, followed by roots and stems. The gene was highly expressed in Escherichia coli BL21 (DE3), and the optimal expression conditions were as follows: isopropyl 尾 -D-thiogalactopyranoside (isopropyl 尾 -D-1-thiogalactopyranoside) was added at 37 鈩，

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