大豆PM2蛋白及其结构域可提高烟草耐盐性

深圳市微生物基因工程重点实验室已利用大肠杆菌异源表达体系证明了大豆PM2蛋白（LEA3）的耐盐功能，并首次证实PM2蛋白序列中的22-氨基酸结构域（PM2B）为一主要耐盐结构域．为在高等植物中进一步验证PM2蛋白及22-氨基酸结构域的耐盐功能，将大豆PM2及PM2B基因克隆至植物表达载体pB1121，并利用农杆菌侵染法转化烟草叶盘，通过PCR扩增法鉴定转基因植株．结果表明PM2及PM2B基因已整合至转基因烟草的基因组DNA中，转化率分别为44．4％和62．5％．与含1．5％（质量分数）NaCl的培养基比较，转基因烟草植株的生长状况和叶片的叶绿素含量，结果表明转PM2和PM2B基因的烟草耐盐性明显高于对照（转化pB1121载体）植株．这说明转PM2和PM2B基因可提高烟草的耐盐能力．可见，PM2蛋白及22-氨基酸结构域在原核和真核细胞中的耐盐保护作用可能是相似的．

Soybean PM2 protein and its 22-mer region enhance salt tolerance of tobacco plants

Late embryogenesis abundant (LEA) proteins accumulate in plant seeds during maturation, in pollen, and in vegetable tissue in response to stress conditions or ABA treatment. Recently, LEA-like proteins were also found in nematode, in avian blood islands and human germinal center B lymphocytes. Most LEA proteins are hydro-philic and rich in glycine. They are predicted to be related with salt tolerance in organisms. But the protective mechanism of LEA protein functioning in salt tolerance is still unclear. According to the conserved motif and the primary structure, LEA proteins were divided into at least six groups. Group3 LEA ( LEA3) proteins are characterized as 11-mer repeating motif of " TAQAAKEKAGE". Soybean PM2 protein (Accession No. : M80664) belongs to LEA3 protein family. Besides six copies of 11 -mer repeating motif, there are six copies of 22-mer repeating motif of " VNKMGEYKDYAAEKAKEGKDAT" in PM2 protein sequence. By searching in Genbank database of NCBI, other six embryonic proteins or LEA3 proteins containing 22-mer repeating motifs were found. In a previous paper, soybean full-length PM2 protein was proved by Liu yu et al. to enhance the salt tolerance of Escherichia coli and the PM2 polypeptide composed of 6 copies of the 22-mer repeating motif was first validated to be a main salt-tolerant domain. In the present paper, in order to test the salt tolerant function of PM2 and 22-mer repeating region (PM2B) in plant, the recombined plasmid containing PM2 or PM2B gene was constructed by using pBI121 vector, respectively. The tobacco leaf discs transformed by Agrobacterium tumefaciens containing recombined plasmid or the pB1121 empty vector. The transgenic plantlet was selected on the MS media supplemented with 1.0 mg/L 6-BA, 0.05 mg/L NAA and 100mg/L kanamycin. The genomic DNA of transgenic tobacco plantlets was isolated, and then used for PCR reaction as template. The pattern analysis of PCR indicate that the soybean PM2 and PM2B gene has been integrated into the genomic DNA of transgenic tobacco plants, respectively. The transformation efficiency is 44. 4% and 62. 5% . On MS growing media containing 1. 5% NaCl, the transgenic tobacco seedlings show better growth performance and higher chlorophyll content of their leaves compared with the non-transgenic plant as control. In summary, the expression of soybean PM2 and PM2B gene enhances salt tolerance of transgenic tobacco plants as they function in E. coli as described in our previous paper.