Cloning And Functional Analysis Of Drought-related Genes TaCP And TaLTP1 In Wheat(Triticum Aestivum L.)

Drought is one of the most important abiotic constraint limiting the yield of wheat (Triticum aestivum L.). Discovering and using the genes involved in drought tolerance in plants are of significance not only on further understanding the molecular mechanism of drought tolerance, but also on improving the drought tolerance of crops via genetic engineering.Cysteine protease (CP), with the catalytic activity that is active mainly to degradate and remove cellular components of the dead cells, is universally involved in the process of programmed cell death (PCD). Lipid transfer protein (LTP) lies widely in cytoplasm of animal, plant and microorganism and takes on lipid transport function. CP and LTP are both induced by many abiotic stresses, such as drought, salt, low temperature etc.Many EST clones representing candidate genes important for drought tolerance have been obtained by screening cDNA libraries constructed with water stressed seedling of wheat. With Hanxuan 10, an elite wheat cultivar with drought tolerance, we have cloned wheat cysteine protease gene TaCP and lipid transfer protein gene TaLTPl. Their expression patterns in the time course of water stress were analyzed through northern blot. TaCP and TaLTPl were respectively transformed into Arabidopsis thaliana and their functions in drought tolerance were investigated.The results of the present research are as follows:1. Twenty-three cDNA clones from two cDNA libraries, which were constructed with wheat seedlings treated with PEG-6000 solution for 24h and 48h, were screened by Reverse Northern Blot. A total of 13 up-regulated ESTs and 3 down-regulated ESTs have been identified, and the candidate clones of No. WSI-V-F10 and No. WSI-I-L10 have been validated by Northern Blot.2. Using No. WSI-V-F10 clone from 24h cDNA library as a query probe, wheat cysteine proteinase gene was cloned by RACE method and named as TaCP (Genbank accession number: AY841792), and a patent application for TaCP has been filed (Application number: 200410088515.8; Publicity number: CN 1609209A).3. Based on the sequence of No.WSI-I-L10 clone from 24h cDNA library, wheat lipid transfer protein gene TaLTPl was cloned by in silico cloning and RT-PCR methods.4. Northern analysis showed that both TaCP and TaLTPl were up-regulated by water stress in wheat seedlings, but the expression levels varied during the time course of stress treatment.5. Plant expression vectors for TaCP and TaLTPl have been constructed. Transgenic Arabidopsis thaliana plants harboring TaCP, TaLTPl and empty vector itself, respectively, were generated through agrobacterium-mediated transformation via infiltration. T2 seeds for TaCP transgenic plants and the seeds for homozygous lives of both TaLTPl and empty vector control transgenic...