关键词: 烟草, 天冬氨酸蛋白酶, RACE, 实时荧光定量PCR, 马铃薯Y病毒

Abstract:

【Objective】 The objective of this study was to screen and clone PVY infected resistance-related genes, and to analyze their relative expression quantity, explore the molecular mechanism of PVY resistance and provide a theoretical foundation for PVY-resistance tobacco (Nicotiana tabacum) breeding. 【Method】 A middle gene fragment of up-regulated expression (Ratio＞2) was screened from the tobacco leaf suppression subtractive hybridization (SSH) library induced by PVY using SSH and cDNA chip technologies, its full-length cDNA sequence was cloned through rapid amplification of cDNA ends (RACE), and its relative expression quantity in different infection times was analyzed by real-time quantitative PCR. 【Result】 A middle gene fragment of 595 bp was screened from tobacco leaves induced by PVY, and a full-length tobacco aspartic protease cDNA of 1 770 bp was cloned by RACE and named Ntasp (GenBank accession no.GU144571), which encoded a protein of 506 amino acids. Sequence alignment indicated that Ntasp revealed a high degree of similarity with other members of plant aspartic proteases and had a typical domain characteristic. The results of real-time quantitative PCR showed that expressions of Ntasp transcripts were up-regulated in tobacco leaves infected by PVY in early stage. 【Conclusion】 A tobacco aspartic protease gene Ntasp was cloned and induced by PVY infection.

Key words: Nicotiana tabacum, aspartic protease, RACE, real-time quantitative PCR, Potato virus Y