毛头鬼伞真菌具有抗烟草花叶病毒活性的y3基因的克隆及对烟草的转化

Cloning of y3 gene encoding a tobacco mosaic virus inhibitor from Coprinus comatus and transformation to Nicotiana tabacum

摘要:

摘要：【目的】 y3基因的表达产物Y3蛋白具有抑制TMV的活性。本文拟克隆y3基因cDNA 全长序列，构建植物表达载体并转化植物，以进一步了解y3基因的功能及它在活体中的表达状况。【方法】首先用试剂盒5′ -Full RACE Core Set（TaKaRa）进行5′ -RACE扩增y3基因cDNA的5′末端未知序列，随后用RT-PCR扩增毛头鬼伞（Coprinus comatus）总RNA以克隆y3基因cDNA全长序列，并用它构建植物表达载体，通过农杆菌介导法转化烟草。【结果】我们得到了y3基因cDNA全长序列。y3基因由534个碱基对构成，含有1个开放阅读框（ORF）。此ORF编码130个氨基酸残基。在本实验中得到的cDNA序列和由此推导的氨基酸序列都表现出了与先前发表的y3基因的部分对应片断具有高度的相似性，均达94%。利用y3基因全长序列信息，我们克隆了y3基因cDNA 序列，并用它与pBI121和pCAMBIA1301构建了植物表达载体pCAMBIA1301-y3。新构建的载体主要由pCAMBIA1301构成，并在它的多克隆位点（MCS）上插入了y3基因序列和来自pBI121的CaMV 35S启动子和NOS末端。这种植物表达载体pCAMBIA1301-y3用于烟草转化, 获得的转基因植株经PCR，RT-PCR和Northern杂交分析证实y3基因已经整合到烟草基因组中，并在其中得到表达。对TMV的抗性鉴定表明，转基因烟草表现出一定的抗病毒活性。【结论】y3基因在转基因烟草植株中得到整合与表达，并表现出一定抗病毒活性。y3基因全长序列的获得和对烟草（Nicotiana tabacum）的成功转化为y3基因的进一步研究奠定了基础。

Abstract:

Abstract: 【Objective】Since protein Y3 encoded by gene y3 reduced TMV activity, we intended to clone the full length of gene y3 in order to study gene y3 and its inhibitory function against TMV in in vivo conditions.【Methods】We amplified the unknown 5′- terminal cDNA sequence of gene y3 with 5′-RACE and using 5′- Full RACE Core Set (TaKaRa), and then the full length of gene y3 was obtained by RT-PCR amplification of total RNA of Coprinus comatus. The expression plasmid of y3 was constructed on the basis of gene y3 and transformed into Nicotiana tabacum via agrobacterium- mediation. 【Results】We obtained the full length of y3 successfully. It consisted of 534 bps including one ORF that encodes 130 amino acid residues. The cDNA sequence and its deduced amino acid sequence, which were obtained in this study, showed high similarity (up to 94%) to known fragment of gene y3 sequence. According to the full sequence, we cloned gene y3 and constructed the expression plasmids. The plasmids were constructed via inserting gene y3 sequence, CaMV 35S promoter, and NOS terminator, which were obtained from pBI121, at the MCS of pCAMBIA1301. The plasmids were used for gene transformation, resulting in transgenic Nicotiana tabacum plantlets. Gene y3 transcription was characterized by Northern blot analysis. Inoculation tests of the transgenic plantlets with TMV demonstrated inhibitory activity against TMV. 【Conclusion】Gene y3 transcription was expressed in transgenic tobacco plants. Gene y3 demonstrated some inhibitory function against TMV in vivo. The full length of gene y3 obtained in this study and its expression in Nicotiana tabacum plantlets could provide background information and benefits for future study on gene y3.