过表达海洋微生物宏基因组MbCSP提高转基因拟南芥的抗旱和耐寒性

干旱和低温是影响农作物生长发育的重要因素，培育转基因作物是解决此问题的有效途径。冷激蛋白（Cold Shock Proteins，CSPs）是一类高度保守的核酸结合蛋白，参与非生物胁迫应答等细胞生理活动，转CSP基因可增强作物抗逆能力。以海洋微生物宏基因组DNA为模板，采用锚定PCR的方法克隆得到了MbCSP基因，其ORF为216 bp，编码一个由71个氨基酸构成的蛋白；对其进行同源性分析，显示该氨基酸序列与EcCSPG、EcCSPA（大肠杆菌 Escherichia coli），BsCspB（枯草芽孢杆菌Bacillus subtilis）和BcCspA（蜡样芽孢杆菌 Bacillus cereus）等冷休克蛋白氨基酸序列同源性在60%~90%；对该氨基酸序列进行多重序列比对和系统发育树分析，结果发现MbCSP蛋白包含RNP1（KGFGFI）和RNP2（VFVHF）等CSP蛋白经典的保守结构域，其与EcCspG（大肠杆菌）和CmCspG、CmCspB（堆肥宏基因组）等生物的冷休克蛋白亲缘关系较近。为进一步探讨冷休克蛋白MbCSP的功能，构建了植物表达载体pTF101-MbCSP，采用花序浸染法转化拟南芥，经过除草剂筛选和PCR检测，获得转基因植株。进行半定量RT-PCR分析，选择表达量最高的阳性株系进行后续的生理检测。结果表明：在干旱胁迫及低温胁迫下，转基因拟南芥的生长状况明显优于野生型，其生物量显著高于野生型植株；转基因拟南芥的叶片相对含水量、叶绿素含量和超氧化物歧化酶活性均高于野生型拟南芥，而丙二醛含量则低于野生型拟南芥。上述结果表明，过表达海洋微生物宏基因组MbCSP能够提高转基因拟南芥的抗旱和耐寒能力，为培育转基因作物新品种奠定了基础。

Overexpression of Marine Microbial Metagenomic MbCSP Enhanced Drought and Cold Tolerance of Transgenic Arabidopsis thaliana

Drought and low temperature are important factors affecting the growth and development of crop,genetically modified crop is an effective way to solve this problem.Cold shock proteins(CSPs)are a class of highly conserved nucleic acid binding proteins,which participate in cell physiological activities such as abiotic stress response,and the transfer of CSP gene can enhance the stress tolerance of crop.In this study,MbCSP gene was cloned by anchor PCR with marine microbial metagenomic DNA as template,and ORF was 216bp,encoded 71 amino acids.Homology analysis showed that the amino acid sequence had homology from 60%to 90%with cold shock proteins such as EcCSPG,EcCSPA(Escherichia coli),BsCSPB(Bacillus subtilis)and BcCSPA(Bacillus cereus),respectively.The results of multiple sequence alignment and phylogenetic tree analysis showed that MbCSP protein contained the classical conserved domains of CSP proteins such as RNP1(KGFGFI)and RNP2(VFVHF),and were closely related to the cold shock proteins of EcCSPG(E.coli)and CmCSPA,CmCSPB(compost metagenome).To further explore the function of MbCSP,a plant expression vector pTF101-MbCSP was constructed,and transfered to Arabidopsis thaliana by floral dip method.Transgenic plants were obtained by herbicide screening and PCR.Semi quantitative RT-PCR analysis was carried out,and the positive strain with the highest expression level was selected for subsequent physiological detection.The results showed that the growth of transgenic Arabidopsis plants were significantly better than that of wild-type,and its biomass was significantly higher than that of wild-type,under drought stress and low temperature stress.The relative water content,chlorophyll content and superoxide dismutase activity of transgenic Arabidopsis plants were higher than those of wild-type,while the content of malondialdehyde was lower than that of wild-type.The results showed that overexpression of marine microbial metagenomic MbCSP could improve drought and cold tolerance of transgenic A.thaliana plant,which laid the foundation for cultivating new varieties of transgenic crops.