香蕉cDNA文库构建及镉胁迫耐受基因的初步筛选

Construction of cDNA Library and Preliminarily Screening of Cadmium Tolerant Genes from Banana

为分离香蕉镉胁迫耐受相关基因,以香蕉为材料,用100μmol/L氯化镉处理诱导香蕉根系3 d,利用gateway技术构建香蕉c DNA文库。经检测,文库滴度为2.0×106 CFU/m L,库容量为8.0×106 CFU/m L,插入平均片段大于1 kb,片段分布在500～2 000 bp,重组率为100%。将该文库质粒转入对Cd敏感的酵母突变株ycf1Δ,采用FOX技术(Full-length c DNA over-expression gene hunting system)进行筛选,同时进行酵母互补实验功能验证,获得1个能够恢复ycf1Δ对镉敏感表型的重组质粒,测序分析得到此重组质粒包含的cDNA全长序列是香蕉快速碱化因子(MaRALF)。结果认为此基因为香蕉体内编码Cd耐受的候选基因。本研究为香蕉MaRALF基因挖掘更多的功能及完善香蕉在逆境胁迫环境中的机理提供了理论依据。

In order to isolate the genes related to cadmium stress resistance in banana, using banana as materials, a cDNA library was constructed with 100 μmol/L CdC12 treatment for 3 days by gateway technology. After detec- tion, the average titer and capacity of library were 2.0×10^6 and 8.0×10^6 CFU/mL, respectively, with the average fragment over 1 kb, mainly distributed in 500-2 000 bp. The recombination rate of this library was 100%. The li brary plasmid was transformed into Cd-sensitive yeast mutant ycflA and screened by FOX （Full-length cDNA overexpression gene hunting system）. Meanwhile, functional verification of yeast complementation experiment was carried out to obtain a recombinant plasmid that could recover the sensitivity ofycflA to cadmium. Sequence analyses indicated that cDNA full-length sequence included in the recombinant plasmid was Musa acuminate rapid alkalization factor （MaRA LF）. The results preliminarily indicated that the gene was candidate gene encoding Cd-tolerance in banana. This study might provide a theoretical basis for MaRA LF gene to explore more functions and improve the mechanism of banana in stress environment.