Cloning And Function Analysis Of Three Articles Of Drought Resistance Related Genes From Grimmia Pilifera

Grimmia pilifera is belongs to the Grimmiaceae Grimmia, it is a sort of typical drought-tolerant moss as an excellent anti-drought genetics resource. In order to study the Grimmia pilifera molecular mechanism of its drought tolerance, three genes associated with drought were selected from Grimmia pilifera drought stress cDNA library: PS? reaction center protein gene(GpPsbR),reduction of strss response gene(GpOsmC),ubiquitin carboxy terminal hydrolytic enzymes(GpUCH). We obtained full length cDNA sequence of GpOsmC, GpUCH through 3?-RACE clone and analyzed these three genes through bioinformatics methods. Quantitative real-time PCR analysis showed that the expression of three genes was induced in different situations and constructed expression vectors. The three genes were transformed into tobacco genome by agrobacterium-mediated transformation method and measured physiological and biochemical indexes. The main results obtained were as follows:(1) The result of bioinformatics shows that the full length of GpPsbR gene is 423 bp which encoded 47 amino acids. Blastn analysis from NCBI showed that the amino acid sequence of the protein encoded by GpPsbR shares a high similarity with physcomitrella patens of 86% identity. The phylogenetic analysis indicated that the genetic distance between GpPsbR and PsbR from physcomitrella patens is the most closed; the full length of GpOsmC gene is 658 bp which encoded 159 amino acids. Blastn analysis from NCBI showed that the amino acid sequence of the protein encoded by GpOsmC shares a high similarity with physcomitrella patens of 80% identity; the full length of GpUCH gene is 871 bp which encoded 236 amino acids. Blastn analysis on NCBI showed that the amino acid sequence of the protein encoding by GpUCH shares a high similarity with physcomitrella patens of 81% identity. The phylogenetic analysis indicated that the genetic distance between GpUCH and UCH from physcomitrella patens is the most closed.(2) Quantitative real-time PCR analysis showed that the expression of these genes was induced in both rehydration and dehydration progress. Under different conditions the expression of these genes were obviously various. The similar expression level was found in these genes and the highest expression level was found in 60 min after rehydration, the expression was declined in six hour after rehydration. During the rehydration, expression of the three genes raised steadily. During dehydration, the expression level were restrained earlily and then sharply raised after 30 min dehydration. The results suggest that thses genes may play an important role in drought stress.(3) At the foundation of pRI101-AN expression vector, the three genes was transformed into tobacco genome by agrobacterium-mediated transformation method. The PCR result showed that these genes were integrated into the genome of tobacco. Measured physiological indexes about transgenic T1 tobacco plants showed that transgenic tobacco exhibited more tolerance to drought stress.