Cloning And Functional Investigation Of Salt Stress Response Genes TaACO1 And TaSTPK Of Wheat (Triticum Aestivum L.)

Salinity stress is one of the major negative abiotic stresses for crop yields, and soil salinization area increasing continuously with the influences of climate change and pollution which impairs the production of crops. Wheat is one of the most important crops for human in the world. Consequently, it is very important to investigate wheat salt tolerant mechanisms, isolate salt responsive genes and breed salt tolerant wheat strains to expand the wheat planting area, increase the unit area production, ensure food security.Previous study got a suppression subtractive hybridization cDNA library expression chip in salt treated plant roots from both Shanrong No.3 and Jinan 177. A serial of candidate salt tolerant genes were found after functional prediction on differential expression genes. This work focuses on the cloning and functional study of two down-regulated genes under salt treatment, TaACO1 and TaSTPK. With the aid of molecular, biochemical and physiological experiments, this work has confirmed the subcellular localization, expression regulation with different abiotic stresses of the two genes, and verified the biochemical and physiological function of recombinant proteins, furthermore, by using overexpression transgenic lines, their physiological function has been further confirmed. The main results are summarized as follows:1. Cloning and functional analyses of TaACO1ORF and genomic sequences were cloned from Shanrong No.3 and Jinan 177. The ORF of TaACO1 contains 942 bp and is composed with 4 exons and 3 introns, and its sequence is identical in Shanrong No.3 and Jinan 177, indicating the TaACO1 in Shanrong No.3 is from Jinan 177. The sequence similarity was compared between TaACO1 and its homologous genes in other specieses, and 80% for Os11g0186900 in rice, 60% for two ACO genes from maize, about 40% for some ACO genes in Arabidopsis and tomato, respectively. The expression of TaACO1 at transcription level was down-regulated when Shanrong No.3 and Jinan 177 were treated with 200 mM NaCl or 100 mM H2O2, and which is comparable with the data from the gene chip. In contrast, it was up-regulated when plants were treated with 100μM ACC, the well known substrate of ACO, and which suggest the substrate induced expression of TaACO1 regulation mechnism. With the aid of TaACO1-GFP, we found that the TaACO1 subcellullarly expressed in nuclei. For biochemical study of the TaACO1, prokaryotic expression vector was constructed and transformed into E.coli BL21 (DE3), and the recombinant protein was found to be expressed in both supernatant and inclusionbody. After purified from the supernatant, the recombinant protein was proved with ACO activity in catalyzing ACC into ethylene, and which showed that TaACO1 is a functional ACC oxidase in wheat. Further physiological function of TaACO1 was analyzed in Arabidopsis, and over-expression transgenic lines were with longer hypocotyl than wild type backgroud and empty vector transgenic control line, since low concentration of ethylene could induce hypotyl growth, so over-expression of TaACO1 may led to ethylene procuction.2. Cloning and functional study of TaSTPKTaSTPK is predicted encoding a Ser/Thr protein kinase, and it was cloned from Shanrong No.3 and Jinan 177 respectively. The ORF of it contains 1485 bp and the genomic sequence consisted of 6 exons and 5 introns. And the gene cloned from Shanrong No.3 and Jinan 177 is with 5 bp differences, and which could influence 4 amino acids, and the sequence of Shanrong No.3 was selected for further biochemical and physiological experiments. When treated with 200 mM NaCl or 100 mM H₂O₂, the expression of TaSTPK in Shanrong No.3 and Jinan 177 was down-regulated in roots, and is comparable with the data in gene chip. Subcellullar localization result showed that TaSTPK is expresseded in both nuclei and cytoplasm. When heterogeneously expressed in E.coli BL21 (DE3), the recombinant protein was mostly expressed in inclusion body. The seeds of TaSTPK overexpressing Arabidopsis lines germinate earlier and show higher germination rate than empty vector transgenic line under NaCl treatment, indicating TaSTPK1 is an important stress responsive gene for seed germination.Conclusionaly, the work in this thesis successfully cloned full length of the two stress responsive genes, TaACO1 and TaSTPK1, and results clarifyied that these two genes are salt responsible and are involved in plant-stress interactions. Further experiments demonstrated their subcellular expression, biochemical character of the recombinant proteins, and physiological function of these genes when heterogeneously over-expressed in Arabdiopsis. This work supplied direct evidences and primary mechanisms on the involvement of TaACO1 and TaSTPK1 in plant aboitic stress responses, and this will be helpful for the understanding of salinity-tolerant mechanism of Shanrong No.3 and supplying useful gene candidates for breeding stress-tolerant crop strains by using transgenic methods.3. Identification and analysis of Ca²⁺ channel on Vicia faba guard cell plasma membrane Here, we employed Ca²⁺ instead of Ba2+ in recording Ca²⁺-permeable channels on Vicia faba guard cell plasma membrane to mimic physiological solute conditions inside guard cells more accurately. Our data suggest that a type of voltage dependent Ca²⁺ channels also mediate K⁺ efflux at depolarization voltages. In addition, a new type of high-conductance channels with 5-fold greater Ca²⁺ conductance and 18-fold greater outward K⁺ conductance than normal channels was also found. This work is very helpful for understanding of new aspects of guard cell Ca²⁺ channels, and will be useful for further searching and identification of Ca²⁺ channel encoding genes.