Construction And Screening Of A NaCl-induced CDNA Library And Functional Analysis Of Salt-Tolerance Genes From Gossypium Hirsutum

Cotton is one of the most important economic crops in our country. People are always interested in identifying and developing new cotton breed to make it can be widely cultivated in saline-alkali soil. Recently, a great achievement has been obtained. However, the salt tolerance ability of cotton is still very limited. So identification of the corresponding salt-tolerant cotton will have important implications for agriculture. The transgenic technology provides us with new ways to improve the plants tolerance by transferring useful genes to different plants. In this experiment, a NaCl-induced cDNA library of cotton seedlings was constructed in order to isolate more salt-tolerance genes from cotton. The library was validated to be successful by titering. 17 salt-induced clones were identified after screening the cDNA Library. Clone 5, 17, 30, and 21 were selected to studied respectively.Metallothioneins (MTs) are defined as a class of proteins with the characteristics of low molecular weight, high cysteine (Cys) residue content and metal-binding ability. They have been widely found in animals, plants, fungi and cyanobacteria. Plant MT-like proteins are divided into three major classes (MT-I, MT-II, MT-III ), which are distinguishable on the basis of the distribution of cysteine residues in their amino acid sequences. Clone 5 and 17 belong to class MT-I. The stucture and function of Clone 5 and 17 were studied. The main results are as follows:1. Clone 5 and 17 have high sequence homology with each other, the identity of Nucleotide sequences is 91.15%, and identity of the amino acid residues sequences can reach 85.94%. These two clones showed high homology with metallothein-like genes from various plants species, such as Rubus idaeus, Carica papaya, Vitis vinifera, Actinidia chinensis etc. The phylogenetic analysis of these proteins suggested that those proteins all belong to the same protein family. Take together, our results indicated that clone 5 and 17 encode two MT-I proteins, which were designated as GhMT1 and GhMT2.2. Northern blot analysis indicated that the mRNA accumulation of GhMT1 was induced by salt stress (300mM NaCl), and the mRNA level kept increasing in the following 12 hours. The expression of GhMT1 was also up-regulated by 300μM CuSO4, 300μM ZnSO4, low temperature (4℃), drought (25% PEG), ABA (abscisic acid) and ethylene, suggesting that MT-I in cotton was not a specific salt stress response gene family but an abiotic stress response gene family. This result also suggested that GhMT1 might play a role in signal transduction of ABA and ethylene.3. Northern blot analysis also showed that the expression was repressed by N-Acetyl-L-cysteine (NAC) when treated by NaCl, low temperature (4℃) or drought (25%PEG). Analysis of H2O2 level showed that NAC repress the H2O2 level increase in above stress. .Therefore, reactive oxygen species (ROS) is essential for activating the GhMT1 and other member expression of GhMT I family under abiotic stress.4. The transgenic tobacco plants overexpressing GhMT1 and GhMT2 grew better than wild type tobacco in 200 and 300 mM NaCl solution. The transgenic tobacoo plants also grew better under low temperature (4℃), drought (25%PEG) condition. Northern blot analysis showed that the GhMT1 and GhMT2 genes had higher transcription levels in transgenic tobacco plants, while there was no GhMT1 and GhMT2 expression in wild-type tobacco plants.5. The increased SOD activities were observed in transgenic tobacco plants and wild type tobacco plants after various abiotic treatments (NaCl, 4℃, 25% PEG). However, the SOD activities in transgenic plants was higher than in wild type plants, indicating that over-expression of GhMT1 could increase the activity of SOD, which maybe the reason that GhMT1 and its members could improve abiotic stress tolerance of cotton.6. To test the binding of GhMT1 to metal Zn2+, we expressed GhMT1 in E. coli. The results showed that the purified GhMT1 could bind Zn2+ions in vitro ,and the binding ability is no more than five Zn2+ions per MT molecule.Peptidyl prolyl cis/trans isomerases (PPIases, EC 5.2.1.8) play a role in protein folding and function by twisting the backbone of target proteins. PPIases are abundantly expressed in virtually all organisms and all cellular compartments .These ubiquitous proteins can be divided into three families, cyclophilins, FK506-binding proteins (FKBP), and parvulins. PPIases may also act as chaperones either in a PPIase domain-dependent or-independent manner, and they may have essential overlapping functions with other PPIases and chaperones. GhPPI of clone 30 belong to parvulins. The stucture and function of GhPPI were studied. The main results are as follows:1. Clone 30 has high sequence homology with peptidyl-prolyl cis-trans isomerase PPIC-type family (Pin4) from Arabidopsis Thaliana , Oryza sativa and Mus musculus etc. The phylogenetic analysis of these proteins suggested those proteins belong to parvulins family. Sequence alignment showed that GhPPI is more similar to hPar14 or hPar17 type parvulin with 55% identity to hPar14(also known as PIN4/EPVH/hPar14) and hPar17. Taked together, our results indicated that clone 30 encodes peptidyl-prolyl cis-trans isomerase, which is more similar to hPar17, and were designated as GhPPI.2. Subcellular localization of the transiently expressed pBI121-GhPPI-GFP fusion protein in onion epidermal cells was done, the results show GhPPI-GFP fluorescence is concentrated to the nucleus or to the chromosome.3. To assay activity of GhPPI PPIase, expression vector of pET30 a (+)-GhPPI was Constructed and expression of pET30 a (+)-GhPPI was induced with IPTG. The results showed that the purified GhPPI has low activity, these indiced the GhPPI is a new peptidyl-prolyl cis-trans isomerase.4. Northern blot analysis indicated that the mRNA accumulation of GhPPI was induced by salt stress (300mM NaCl), and the mRNA level kept increasing in the following 12 hours. The result suggested that GhPPI might play a role to improved abiotic stress tolerance of cotton.5. GhPPI share a similar C-terminal PPIase domain and an similar N-terminal Lys-rich domain with hPar14 and hPar17. The basic N-terminal domain of hPar14 or hPar17 is responsible for the phosphorylation regulated partition between cytosol and nucleus and its affinity for DNA binding. So GhPPI may have regulatory functions as that in hPar14 and hPar17. It may be better that AtPIN, as GhPPI, also be classified into hPar17 type than into hPar14 before.6. AtPPI was cloned and over expression vector, antisense expression vector and RNAi pFGC5941 vector were constructed. The respective transgenic Arabidopsis plants were identified. 14 days old of Seedlings of transgenic RNAi Arabidopsis were grown stronger and faster than wild type, this is an interesting phenomena.The sequence and expression analysis, function identification of Clone 21 were also studied. The main results are as follows:1. Clone 21 has high sequence homology with ultraviolet-B-repressible protein. The phylogenetic analysis of these proteins suggested those proteins belong to ultraviolet- B- repressible protein family. Clone 21 has also high sequence homology with photosystem II subunit X(Psb X). Take together, our results indicated that clone 21 encodes putative utative ultraviolet-B-repressible protein, which were designated as GhUVB1.2. Subcellular localization of the transiently expressed pBI121-GhUVB1-GFP fusion protein in onion epidermal cells shows GhPPI-GFP fluorescence is concentrated to the membrane of cell and karyotheca repectively.3. Over expression of GhUVB1 in the transgenic Arabidopsis plants show that the T0 transgenic Arabidopsis plants were grown slowly and flower lag of 7days. The phenomic lag of transgenic plant by over expression GhUVB1 in Arabidopsis plants show was smilar to cryptochrome CRY1. Take together, these results indicated that GhUVB1 maybe is closed relative with cryptochrome.