The Cloning And Functional Analysis Of Stress Tolerance Related Genes From Thellungiella Halophila

Drought, high salinity and low temperature are the most common abiotic stresses that affect plant growth and crop yield. Plants respond and adapt to these environmental challenges with a number of physiological and biochemical changes The expression of many genes is induced or repressed when a plant is subjected to abiotic stresses. These genes could be categorized into two groups. The first group genes encode functional proteins, which are directly associated with protection of plants from abiotic stress, such as late embryogenesis abundant (LEA) proteins, enzymes for the synthesis of sugars, proline, and glycine betaine. The other group genes encode regulatory proteins, which regulate signal transduction and stress-inducible genes expression, including various kinds of transcription factors and protein kinases. Many genes are induced by more than one kind of stresses and participate in multiple signaling pathways which constitute a signal network, so there may be cross-talk between these signaling pathways.CBF3(C-repeat binding factor3) is a kind of transcription factors, mainly involved in the regulation of plant response to low temperature stress. Its overexpression can increase cold stress tolerance in plants. CDPKs (Calcium-Dependent Protein Kinases) constitute a large family of serine/threonine protein kinases that involved in a wide array of signaling pathways in plant adaptation to various environmental stresses including drought, salt and low temperature. SRK2C (SNF1-related kinasel) is also a kind of serine/threonine protein kinase, its overexpression could significantly increase the drought tolerance of Arabidopsis plants. P5CS (△1-pyrroline-5-carboxylate synthetase) and GolS2(galactinol synthase) are respectively the key enzymes involved in the biosynthesis of proline and RFOs (raffinose family oligosaccharides), which as osmoprotectants, can improve plant tolerance to osmotic stress. Previous studies found that CBF3, CDPK6, GolS2, P5CS2and SRK2C play important roles in plant adaptation to abiotic stresses. In this study, the full-length cDNAs of these five genes from Thellungiella Halophila were cloned and their sequence were compared with the corresponding genes sequence from Arabidpsis published on the NCB1website. The result showed that the sequences of CBF3, CDPK6and SRK2C are the same with that of Arabidopsis genes The sequences of GolS2had a single base change but did not cause changes in the amino acid level. However, one base change in P5CS2gene sequence resulted in changes of alanine (81) into valine, but they both belong to aliphatic amino acid. To further study the function of the five genes, two RNA inference structures of every target gene were designed through multiple sequence alignment to lower the expressing of target genes. Then the plant expression vectors were constructed, and transformed into Arabuhpsis, and finally the homozygous transgenic lines were obtained. In addition, three mutants for every target gene were identified as the control.Phenotypic comparison of the transgenic plants, mutants and the wild-type plants under the natural conditions suggested that wild-type plants grew slower than othersOn the media supplemented with different concentrations of ABA the seed germination rates from different lines were decreased, and the germination rates declined with the increase of ABA concentrations, however, there was no significant difference among the transgenic lines, mutant lines and wild-type. When the concentration of ABA was2.0μ.M, the seedlings grew slowly, and no significant differences existed among different lines. This meant that the lines were sensitive to ABA.When the seeds of different lines germinated on the media supplemented with different salt concentrations, the germination rate of the wild-type plants gradually higher than the transgenic lines and mutant lines with the salt concentration increased, especially CBF3RNAi lines and GolS2RNAi lines of transgenic Arabidopsis. Under the100mM NaCl conditions, most of the transgenic plants etiolated more seriously than wild-type plants, and the root length of the RNAi lines was shorter than that of the wide type. The salt tolerance ot CDPK6, GolS2and P5CS2RNAi lines declined seriously.On the media supplemented with different mannitol concentrations the germination rate of the wild-type plants were higher than that of the transgenic lines and mutant lines. When the mannitol concentration increased to300mM, CBF3RNAi lines and GolS2RNAi lines of Arabidopsis showed low seed germination rates. Under the200mM mannitol conditions, most of the transgenic plant leaves were smaller and narrower than that of wild-type plants, and the root length is shorter. The drought tolerance of GolS2and P5CS2RNAi lines declined more seriously.Comparison of the seed germination rates under different H2O2concentrations revealed that the germination rates decreased gradually with the increase of H2O2concentrations, but there is no significant differences among the transgenic lines, mutant lines and wild-type.It’s concluded that these five genes had no obvious role in the resistance to ABA or H2O2stress, but CDPK6, GolS2and P5CS2play important roles in plant adaptation to salt stress and osmotic (drought) stress.