Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East West Road, Room 218, Honolulu, HI 96822, USA.
Plant Physiol Biochem. 2010 Apr;48(4):273-8. doi: 10.1016/j.plaphy.2010.01.005. Epub 2010 Jan 22.
The use of the tree-legume Leucaena leucocephala (leucaena), which contains high levels of proteins in its foliage, is limited due to the presence of the toxic free amino acid mimosine. The goal of this research was to develop transgenic leucaena with reduced mimosine content. Two genes, pydA and pydB, encoding a meta-cleavage dioxygenase (EC 1.13.11.2) and a pyruvate hydrolase (EC 3.7.1.6), respectively, from the mimosine-degrading leucaena symbiont Rhizobium sp. strain TAL1145, were used to transform leucaena. These bacterial genes were sequence-optimized for expression in leucaena and cloned into the plant binary vector pCAMBIA3201 for Agrobacterium tumefaciens-mediated transformation. Using immature zygotic embryos as the start explant material, six pydA and three pydB transgenic lines were developed. The presence and expression of the bacterial genes in the transgenic lines were verified by PCR, reverse transcriptase PCR, and Southern analyses. HPLC analyses of the transgenic plants determined that the mimosine contents of the pydA-expressing lines were reduced up to 22.5% in comparison to the wild-type. No significant reduction in mimosine content was observed in the pydB-expressing lines. This is the first example of using a gene from a bacterial symbiont to reduce the toxicity of a tree-legume.
利用树叶中含有高蛋白的豆科植物银合欢（Leucaena leucocephala）受到了限制，因为其含有有毒的游离氨基酸——含羞草氨酸。本研究的目的是开发含羞草氨酸含量降低的转基因银合欢。分别来自于银合欢共生根瘤菌 Rhizobium sp. strain TAL1145 的两个基因，编码一个间裂加双氧酶（EC 1.13.11.2）和一个丙酮酸水解酶（EC 3.7.1.6），pydA 和 pydB，被用于转化银合欢。这些细菌基因经过序列优化，以便在银合欢中表达，并被克隆到植物双元载体 pCAMBIA3201 中，用于农杆菌介导的转化。使用未成熟的合子胚作为起始外植体材料，开发了 6 个 pydA 和 3 个 pydB 转基因系。通过 PCR、反转录 PCR 和 Southern 分析验证了这些转基因系中细菌基因的存在和表达。HPLC 分析表明，与野生型相比，pydA 表达系的含羞草氨酸含量降低了 22.5%。在 pydB 表达系中未观察到含羞草氨酸含量的显著降低。这是首例利用细菌共生体中的基因降低豆科植物毒性的例子。
