Enhancement of freezing tolerance of plants based on studies of mechanisms of low-temperature-inducible antioxidative enzymes from Chlorella

nadph依赖性硫氧还蛋白还原酶定位于小球藻叶绿体（CvNtrC），在转录和翻译水平上被证实是低温诱导的。通过体外下拉法分离小球藻中与CvNtrC作用的过氧化物还氧蛋白（CvPrx），并通过n端氨基酸测序对其进行鉴定。其他研究人员克隆了编码CvPrx的基因，并在大肠杆菌中制备了其重组成熟蛋白（mCvPrx）。我们从研究者那里获得了CvPRX基因和mCvPrx蛋白。我们还在大肠杆菌中制备了成熟的重组CvNtrC （mCvNtrC）蛋白。两种蛋白协同表现出抗过氧化氢和过氧化物的过氧化物酶活性。此外，我们用mCvNTRC和/或mCvPRX基因转化了酿酒酵母。mCvNTRC或mCvNTRC/mCvPRX基因的表达增强了酿酒酵母对冷冻、氧化和热胁迫的耐受性，表明这两个基因在抗逆性中起重要作用。为了制备NTRC基因敲除菌株，我们开发了对普通小球藻C-27的转化方法。此外，我们还利用花浸法将这些基因导入拟南芥中。

NADPH-dependent thioredoxin reductase, which is localized in chloroplast, from Chlorella (CvNtrC) was confirmed to be low-temperature-inducible at transcriptional and translational levels. Peroxiredoxin (CvPrx), which works with CvNtrC in Chlorella, was isolated by in vitro pull-down assay and identified by N-terminal amino acid sequencing. The gene encoding CvPrx was cloned and its recombinant mature protein (mCvPrx) was produced in Escherichia coli by other researchers. We obtained the CvPRX gene and mCvPrx protein from the researchers. We also produced recombinant mature CvNtrC (mCvNtrC) protein in E. coli. The two proteins coordinately showed peroxidase activities against H_2O_2 and peroxides. Furtheremore, we transformed Saccharomyces cerevisiae with mCvNTRC and/or mCvPRX genes. Expression of mCvNTRC or mCvNTRC/mCvPRX led to enhancement of S.cerevisiae against freezing-, oxidative-, and heat-stress tolerances, suggesting that these two genes play important roles in stress tolerance. To make a knock-out strain of Chlorella for NTRC gene, we developed the transformation method for C.vulgaris C-27. Furthermore, we introduced the genes into Arabidopsis thaliana with the floral dip method.