Cloning And Functional Analysis Of Salt-tolerance Related Genes CDNA In Wheat

Large area of saline-alkali land exists in the world, high density salt stress lead to the plant growth retarded seriously, even led to the death. The crops planted in the saline-alkali cultivated land often occured a wide range underproduction. In recent years, to transfer the salt-tolerance genes into the aim crops directly had been the most effective means of salt-tolerance crops breeding. It is primary to get the effective salt-tolerance genes in the salt-tolerance crops genetic engineering. The purpose of this thesis is just continue to clone the whole length cDNA of wheat salt-tolerance relevant genes on the basis of formers' job, and to study preliminarily their functions. Hope to make some beneficial exploration for the salt-tolerance genetic engineering of wheat.The series of near isogenic lines (NILs) were abtained from the offspring of wheat mutant. Firstly, the seedling stage salt-tolerance of these NILs was researched by detecting seed activity index, relative root longth, permeability of plasmamembrane (PPM) and the activity of SOD enzyme. The results are basically coincident with the testing result of the salt pond. So it's confirmed that the salt-tolerance ability of the salt-tolerance line RH8706-49 and salt-sensitive line H8706-34 is also difference in seedling stage, these two lines can be regarded as the experiment material in the following study.The 26 cDNA sequences which abtained by cDNA-AFLP were detected by reverse Northern. The results showed that only 6 cDNA sequences appeared positive signal, the other 20 cDNA sequences didn't found clear hybrid signal. According the information of NCBI-BLAST, we selected the No. 23 and 88 as the study object to study their whole gene sequences by 3' RACE and 5' RACE method. In addition, when analyzed the other 33 cDNA sequences by NCBI-BLASTn, we found that No. 81 cDNA could obtain the whole 3' sequence and the transcription direction by electronic splicing.1210bp 3 ' sequence of No. 23 cDNA fragment was obtained by 3 ' RACE and electronic splicing. It contains 834 bp open reading frame (ORF), codes 277 amino acids. Predicted by software, the molecular weight of this peptide is 31426.05D, its isoelectric point is 4.85. This peptide contains 66.8% a helix structure, likely has CAM binding regions and Cytochrome P450 cysteine heme binding region. The 216 to 236 amino acid region of this peptide is a more believable transmemberane region. According NCBI-Blast, this amino acid sequence has higher homologies with cytochrome P450 (pfam00067.11) . So the peptideencoded by No. 23 cDNA sequence is primarily considered having calmodulin-dependent Cytochrome P450 activation.No. 81 cDNA's 890bp whole sequence was obtained by 5' RACE. It contains 663 bp ORF, codes 220 amino acids. Predicted by software, the molecular weight of this peptide is 24918.06D, its isoelectric point is 8.3. It contains 44.1% P structure and 38.2% a helix. Detected by software, it hasn't CAM binding regions, so it primarily is confirmed that it's Ca2+ undependant protein. We also found that this peptide have 2 transmemberane region. The 79 to 99 amino acid region of this peptide is more believable. According NCBI-Blast, this amino acid sequence has higher homologies with Nucleoside diphosphate sugar epimerases (WcaG). So the peptide encoded by No. 81 cDNA sequence is primarily considered having WcaG enzyme activation.669bp sequnce information of No. 88 cDNA was obtained by 3' RACE. Utilizing 5' RACE and electron spelling, we obtained the whole length 1958bp cDNA sequnce. It has 1431 bp ORF, codes 476 amino acids, has 288bp 5' non-code region (NCR). Its 5' NCR is according to the wheat EST CK199370. Predicted by the software, No. 88 gene encoded protein's molecular weight is 51455.3D, its isoelectric point is 7.88 It also contains 40.8% 3 structure and 36.8% a structure, and likely includes CAM binding regions, protein kinase regions and protein kinase ATP binding region. According to the software, this protein does not belong to transmembrane protein. By detected in NCBI, the peptide encoded by No. 88 gene has higher homologies with serine/threonine protein kinases (STKc, SPS1), tyrosine kinase (TyrKc), protein kinase (Pkinase). So the peptide encoded by No. 88 cDNA sequence is primarily considered serine/threonine kinase family, and has tyrosine kinase activation and depends on CAM adjusting. On March 8, 2005, the 1670bp whole length cDNA was submitted to NCBI database. It was named TaSTK. Its submitted number was 702708, and land number was AY956328.According to the Northern hybridization of No. 88------TaSTK, it indicates that TaSTK isa NaCl induced gene in RH8706-49 and H8706-34, and its expression in the salt-tolerance line RH8706-49 is more strongerly induced than in the salt-sensitive line H8706-34. At the same time we can observe that the hybridization signals in four kinds of materials are all very weak. This indicates that TaSTK belongs to the low expression gene in the wheat seedling tissue.In order to study the relevant functions of TaSTK gene, we construct the binary express vector pl300: 35S: TaSTK: NOS. Using the method of floral dip and with the screening of...