羊草自交不亲和性相关基因TrxLc的克隆、表达和酶活性分析(英文)

Molecular Cloning, Expression and Activity Assay of Thioredoxin h — A Gene Related to Self incompatibility in Leymus chinensis

羊草是一种重要的禾本科牧草 ,主要分布于亚洲东部地区。硫氧化还原蛋白H (Trxh)是一大类广泛分布于动物、植物和微生物中的小分子蛋白。在一些禾本科植物中 ,一种在成熟花粉中表达的Trxh ,与植物的自交不亲和性有关。本研究克隆了羊草花粉硫氧还蛋白基因 (TrxLc)的DNA和cDNA序列 ,与其他禾本科植物Trxh的cDNA序列相比 ,它们具有 5 3.5 %～ 5 6 4 %的同源性。Southern杂交显示TrxLc在羊草基因组中是单拷贝。Northern杂交显示TrxLc在羊草根、茎和叶中没有表达 ,在成熟雌蕊和幼小花粉中微量表达 ,但是在成熟花粉中大量表达。在大肠杆菌中表达重组TrxLc并检测了其活性。胰岛素还原反应中得到的Vmax值和Km 值显示 ,TrxLc类似于其他植物的非花粉组织特异表达的Trxh ,但与大肠杆菌的Trxh差异较大。

Leymus chinensis (Trin.) Tzvel., which is widely distributed in East Asia, is an important self incompatible forage species in Poaceae. h type thioredoxins are a family of small proteins that exists throughout the animal, plant, and bacterial kingdoms. Gene for thioredoxin h expressed in mature pollens is related to self incompatibility in some grasses species. In the present study, DNA and cDNA sequences of thioredoxin h ( TrxLc ) gene were cloned from L. chinensis . The cDNA nucleotide sequence of TrxLc had 53.5%-56 4% of homology to thioredoxin h genes that originated from other self incompatible grasses. Using Southern blotting, gene TrxLc was estimated to be one copy in L. chinensis genome. Northern blotting indicated that TrxLc did not express in leaves, roots, stems, and young pistils, but expressed in young pollens and mature pistils. The highest expression level of TrxLc was detected in mature pollens. Gene TrxLc was expressed as recombinant protein in Escherichia coli and tested for thioredoxin activity. In the nonspecific insulin reduction assay, the protein displayed typical thioredoxin activity and had similar values of V max and K m with h type thioredoxins of other plant species, but was not similar with that of E. coli.