关键词: 斑茅, 干旱胁迫, 抑制性消减杂交, 表达序列标签

Abstract: Saccharum arundinaceum Retz, one of the most important wild relatives of Saccharum officinarum L., is a kin species with desirable attributes including excellent vigor and ratooning, adaptability to environmental stresses such as moisture deficits or excesses, and resistance to Pachymetra root rot for sugarcane genetic improvement. Efficient construction of cDNA libraries enriched differentially expressed transcripts is the most important step in biological investigations. Suppression subtractive hybridization (SSH) is a highly effective method for the generation of subtracted cDNA libraries. It is based primarily on a described technique called suppression PCR and combines normalization and subtraction in a single procedure, in which the normalization step equalizes the abundance of cDNAs within the tester population and the subtraction step excludes the common sequences between the tester and driver populations. A SSH library was constructed from the cDNA of the leaves in S. arundinaceum, subjected to water stress as Tester and from cDNA of the leaves in S. arundinaceum, planted in the normal condition as Driver. Titer results indicated that about 30 000 clones were included in the library, and the percentage of recombination was up to 99.2%. Further, 3 500 clones were picked out randomly from the SSH library for cDNA microarray analysis. The size of inserts ranged mainly from 200 bp to 1 000 bp, of which 463 clones were larger than 500 bp and 3 037 clones smaller than 500 bp, with 450 bp on average. After picking up randomly 16 clones for sequencing, the results by using BLAST revealed that there were three drought-stress-associated genes (ATP-NAD kinase, Aldo/keto reductase and zinc finger protein), three novel genes and other 10 function-unknown expressed sequence tags（EST）. Two out of 16 clones, which were termed as EL0149 and EL0145, were chosen for further analysis. It was also confirmed by Northern blot that EL0149 up-regulated in the moderate water stressed leaves, while no hybridization signal of EL0145 was detected in the leaves exposed to either the normal water condition or the moderate water stressed condition. The results suggested that the SSH library constructed from the cDNA of the drought-stressed leaves, when combined with cDNA microarray, should be a good method for analysis of drought-resistance gene network in plants. Saccharum arundinaceum Retz, one of the most important wild relatives of Saccharum officinarum L., is a kin species with desirable attributes including excellent vigor and ratooning, adaptability to environmental stresses such as moisture deficits or excesses, and resistance to Pac

Key words: Saccharum arundinaceum Retz, Drought stress, Suppression subtractive hybridization (SSH), Expressed sequence tags (EST)