The Functional Analysis Of OsKCC Gene And The Functional Analysis Of Small RNAs In Thellungiella Halophila

Salinity is one of the major abiotic stress in the world. Cation transport and homeostasis are issues of special significance in plant biology. In recent years, potassium (K~+) and sodium (Na~+) transporters have been studied extensively because of their roles in both plant nutrition and salt tolerance. It is known that acclimatization of plants to salt stress also requires appropriate regulation of chloride (Cl~-) homeostasis, not only because of its toxicity when overaccumulated in the plant symplast, but also because Cl~- is the most important counter-anion in most saline soils, altering Na~+ and K~+ availability and distribution within plant tissues and cell compartments. Cl~-, like K~+, is an essential nutrient for higher plants and a major osmotically active solute in the vacuole, implicated in osmoregulation and cell elongation. In animals, the cation–chloride cotransporter (CCC) family is essential for adequate homeostasis of the most abundant electrolytes, K~+, Na~+ and Cl~-, playing key roles in cell ionic and osmotic regulation. Recently, Jose′M. Colmenero-Flores found a CCC gene in Arabidopsis which encodes for a cation–Cl~- cotransporter involved in plant development and ion homeostasis. Up to now, The involvement of Cl~- transport in salt stress or plant nutrition has not been properly investigated at the molecular level in monocotyledonous plants. The major purpose of our experiment is using rice (Oryza sativa) as the research material, studying the function of OsKCC gene, elucidating the relationship between OsKCC gene and Oryza sativa's development and salt tolerance.Abiotic stress is one of the primary causes of crop losses worldwide. Much progress has been made in unraveling the complex stress response mechanisms, particularly in the finding and identification of stress responsive protein-coding genes. In addition to protein coding genes, recently discovered microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have emerged as important players in plant stress respons. Subsequent studies have demonstrated an important functional role for these small RNAs in abiotic stress responses. MiRNAs and siRNAs are small noncoding RNAs that have recently emerged as important regulators of mRNA degradation, translational repression, and chromatin modification. Salt cress (Thellungiella halophila) is a close relative of Arabidopsis and becomes the prospective salinity tolerance model. The major purpose of our experiment is using salt cress as the research material, cloning the small RNAs of T. halophila and studying the functions of stress related small RNAs, elucidating the relationship between these small RNAs and T. halophila's salt tolerance. The main results of this study summarized below:1. The cloning and functional analysis of OsKCC gene1) Blastp analysis according to the sequences of cation–Cl~- cotransporter in animals prove that there are four cation–Cl~- cotransporters in rice. We select a OsKCC gene to study the function of it. The primer was designed according to the sequence of OsKCC gene. The full length of OsKCC gene from rice was cloned using RT-PCR method. The open reading frame of OsKCC gene was 2970 bp. It coded a protein of 989 amino acid residues with calculated molecular weight of 109 KD.2) The detailed analysis of the OsKCC gene and protein sequence characterization was made. OsKCC gene had high sequence similarity with the plant CCC gene such as AtCCC. The structure of the OsKCC protein had high similarity with the KCC protein in anmianls and human. The amino acid sequence of OsKCC had 11 predicted transmenbrane regions.3) The detailed analysis of OsKCC gene expression under different kinds of stresses was carried out. Real-time PCR results showed that the expressing levels of OsKCC gene were higher in roots than in shoots and both of them were induced by NaCl and KCl. The KCl treated OsKCC gene's expressing level were higher than treated by NaCl.4) The full length of OsKCC ORF was constructed into the plant expression vector pROKⅡand transformed into Agrobacterium tumefaciens AGO1. The integrated vector was introduced into rice. The OsKCC transformants were continuously screened on the media with G418 (40mg/L). 32 homozygous T3 overexpressing OsKCC gene lines were obtained. Two homozygous transgenic lines were selected and used for molecular and physiological analysis. PCR and real-time PCR confirmed that the OsKCC gene had been integrated into rice genome and had the normal transcription. The physiological analysis showed that the transgenic and wild type rice hadn't any difference under normal or NaCl and KCl stress conditions.5) We contructed a GFP location vector of OsKCC gene and transformed into Agrobacterium tumefaciens AGL1. The integrated vector was introduced into rice and onion. The detailed analysis of the OsKCC protein location was carried out in rice and onion. The OsKCC protein was located not only in the membrane of onion, but also in the membrane of rice.6) The gene silencing expression vector of pROKⅡ-KCC:Ri was constructed and also transformed into rice. 18 G418 resistance plants were selected. Real-time PCR confirmed the expression level of OsKCC gene in gene silenced plants was distinctly lower than in wild type plants. The analysis of the stress tolerance of gene silenced plants showed that OsKCC gene silencing increased gene silence rice KCl and NaCl sensitivity. The K~+ and Cl~- content in the shoot and root of the gene silenced rice were decreased compare with the wild type under KCl and NaCl stress. The Na~+ content had very little difference between gene silenced and wild type rice.2. The cloning and functional analysis of Thellungiella halophila small RNA1) We constructed a small RNA library of T. halophila which was treated by 300 mM NaCl for 24 hours. Sequencing of the library and subsequent analysis revealed 22 miRNAs belonging to 13 families.2) We constructed miR393, miR398 and miR400 silencing expression vectors. Transformed the three gene silencing vectors into T. halophila using floral dipping method.3) We cloned the DNA between ThSRO5 and ThP5CDH gene. We cloned the 3'UTR of the ThSRO5 and ThP5CDH genes. The 3'UTR of the ThSRO5 and ThP5CDH genes in T. halophila haven't the overlapping region. So the T. halophila haven't the SiRNA which the Arabidopsis have to degrade the At P5CDH gene.4) Under NaCl and other stress treatment, the proline of the T. halophila were improved. The content proline were increased with improving of the treat time and concentration of NaCl. Real-time PCR confirmed the expression level of P5CS gene was increased when treated by NaCl and other treatment such as ABA, proline, PEG and 4℃. Real-time PCR confirmed the expression level of P5CS and SRO5 genes were also increased when treated by NaCl and other treatment such as ABA, proline, PEG and 4℃.The main innovation points of this study were generalized as follows:1 First carrying out the detailed research for the OsKCC gene, proving it was a functional membrane K~+-Cl~- cotransporter gene. K~+ and Cl~- can be transport into the cell through the K~+-Cl~- cotransporter. The detailed expression profile of OsKCC gene under different stresses was demonstrated. The analysis of the stress tolerance of gene silencing plants showed that OsKCC gene silencing increased KCl and NaCl sensitivity of gene silencing rice. The K~+ and Cl~- content in the shoot and root of the gene silenced rice were decreased compare with the wild type under KCl and NaCl stress.2 We constructed a small RNA library of T. halophila which was treated by 300 mM NaCl for 24 hours. Sequencing of the library and subsequent analysis revealed 22 new miRNAs belonging to 13 families. We constructed miR393, miR398 and miR400 silencing expression vectors. Transformed the three silencing vectors into T. halophila using floral dipping method.3 The difference of proline change under stress treatment was caused by the siRNA which can degrade the P5CDH in Arabidopsis. The siRNA haven't existence in T. halophila.