Identification Of Drought Stress Response Genes From Aeluropus Littoralis By Suppression Subtractive Hybridization

Drought is a major abiotic factor that significantly limits growth and production of plant. With the intensification of global water shortage, water-saving irrigation has been unable to face the serious challenges of the drought stress. So these years more and more researchers have taken their attention to genetic engineering breeding. The key of genetic engineering breeding is gene mining. Aeluropus littoralis is perennial halophyte monocotyledon with strong drought and salt tolerance, and can be used as a good material for drought-tolerant gene mining. In this study, forward and reverse suppression subtractive hybridization libraries (SSH) were constructed from the whole plants of Aeluropus littoralis subjected to drought stress. The drought stress was representative at three levels (mild, moderate and severe) evaluated by determing leaf relative water content (RWC) and soil moisture content (SMC). Finally the real-time quantitative RT-PCR (qPCR) was used to test the reliability of library and dig drought-tolerant genes.The main research results were as follows:1) The withholding water was used to simulated drought stress. As a result,15%≤SMC<25%and80%≤RWC<85%was defined as mild drought of withholding water5days,10%≤SMC<15%and70%≤RWC<80%was moderate drought with11days of drought stress, and5%≤SMC<10%and60%≤RWC<70%was severe drought after13days of withholding water.2) A forward and reverse SSH library was constructed. In the forward library there were2700clones and in the reverse library there were2200clones. A total of724unigenes (117contigs and607singletons) were derived from sequence alignment and cluster assembly of1288clones.3) The724unigenes were analysed by BLASTX against NCBI nr, NCBI nt, SWISSPORT and KEGG databases. There were662(91.44%) unigenes function annotated successfully, in which173(23.9%) were matched with the protein of function unknown, while the62(8.56%) surplus were considered as novel genes. And many putative drought-responsive genes were identified which included candidate genes of transcription factors, calmodulin-like proteins, ion transporters, heat-shock proteins, and ubiquitin-proteasome. 4) Eight unigenes were selected randomly from two libraries equally. The results showed that most of the genes from forward library were up-regulated, while the genes from reverse library were down-regulated under the drought stress. So the libraries were constructed successfully. Then qPCR was used to dig drought-response genes. We found that most of the genes were up-regulated more than1fold under the drought stress from the forward library. The most significant gene is F-61, with4.36fold of expression after13days of drought stress.