| Wheat?Triticum aestivum L.?is one of the most important food crops in the world,and high yield and quality is an important goal of wheat breeding.Drought is the most important environmental stress factor to limit wheat production and nucleoredoxin?NRX?is associated with drought resistance.Color is one of the important indexes of wheat flour product evaluation and the activity of lipoxygenase?LOX?is closely related to the quality of wheat flour product.Study the gene associated with drought resistance and flour color can lay the foundation for the breeding of high-yielding and high-quality wheat varieties.The drought-tolerant wheat cultivar Jinmai 47 was used as the experimental material and the comparative genomics methods were used to obtain the cDNA?Complementary DNA,cDNA?and gDNA sequence?genomic DNA,gDNA?of three partial homologous genes of TaNRX1 gene,a new member of wheat thioredoxin?TRX?superfamily gene.And the characteristics of encoding proteins were analyzed and the structure of the gene was identified.Also,the expression analysis under drought stress was conducted and the cis-acting elements of the promoter regions were predicted.And plant over-expression vector was constructed,which can lay the foundation for further gene function identification.The TaLOX gene on QLpx.cass-1AL and TaLOX-B1 locus were detected by SSR marker Xwmc312 and STS markes LOX16 and LOX18 in 180 Ningxia wheats,and the distribution features were analyzed,which can provide the theory for breeding and production of wheat in Ningxia.The main results are as follows:1.Cloning and Sequence Analysis of Partial Homologous Genes of TaNRX1 Gene in Wheat.The cDNA and gDNA sequences of three partial homologous genes of TaNRX1 gene in wheat were obtained by comparative genomics methods.The length of the three cDNA sequences were 1815 bp,1827 bp and 1818 bp,respectively;and the opening reading frame?ORF?were 1731 bp,1743 bp and 1734 bp,respectively;and encoding 576,580 and 577amino acids,respectively.The results of sequence alignment showed that the consistency of the ORF of three sequences and encoded amino acid sequences were 98.09%and 97.93%,respectively.The deduced amino acid sequence contained three TRX-like domains and one zinc-finger structure,and the first and third domains both possess a typical WCG/PPC redox activity site and the second domain is atypical TRX-like domain,belonging to type?NRX.The bioinformatics analysis of encoding proteins of TaNRX1 gene showed that the encoded protein did not have typical hydrophobic region and transmembrane structure,and there was no signal peptide.The secondary structure was?-helix,?-sheet,random curl and extended chain,suggesting that the protein is fat-soluble stable non-secreted protein.The phylogenetic analysis of the different species with high homology to the encoding proteins of TaNRX1 gene in wheat showed that encoding proteins of wheat had the closest genetic relationship with Aegilops tauschii and barley,and farthest with Gossypium raimondii.The length of gDNA sequences?from ATG to TAG?of three partial homologous genes of TaNRX1 gene were 3946,4125 and 3903 bp,respectively.And the three sequences were found in the three genomes of the wheat second homologous groups by blast the existing database information and the results were validated by the nullisome-tetrasome ananlysis of Chinese spring?CS?.So they were named TaNRX1-2A,TaNRX1-2B and TaNRX1-2D,respectively.The TaNRX1 gene was found to contain 4 exons and 3 introns by comparing with cDNA and gDNA sequence and the conservation of exons was higher than introns.2.Expression Analysis of TaNRX1 Gene under Simulated Drought Stress and Cis-acting Elements Prediction of Promoter Regions.In our study,two-leaf seedlings of Jinmai 47 were subjected to simulated drought stress treatment with?0.5 MPa PEG-6000solution?with 1/2 Hoagland's nutrient solution preparation?,and leaves were collected at different time points.The total RNA was extracted,and quantitative real-time reverse transcription PCR?qRT-PCR?were carried out and the leaves of normal water supply were used as control.The results showed that all three partial homologous genes of TaNRX1 gene responsed to drought stress,and showed a similar trend,with 2 peaks.The promoter sequences of TaNRX1 gene was obtained by blasting database and the cis-acting elements of the promoter regions were predicted by bioinformatics software.The results indicate that there were ABRE which involved in the abscisic acid responsiveness and MBS that involved in drought-inducibility,but the numbers of cis-acting elements of three partial homologous genes of TaNRX1 gene were not equal.3.Isolation of TaNRX1-2D gene and Construction of Plant Over-expression Vector of TaNRX1-2D Gene.According to the principle of AS-PCR and the alignment results of cDNA sequences of TaNRX1 gene,the specific primers of D genome were designed,and the sequcing results showed that the amplication product was TaNRX1-2D.Subsequently,according to the homologous recombination strategy,the expression vector PLGY-02 was integrated with TaNRX1-2D gene,and sequcing results showed that the plant overexpression vector of TaNRX1-2D gene was successfully constructed.4.Detection and Distribution Analysis of TaLOX Genes in Ningxia Wheat.The distribution characteristics of TaLOX gene at QLpx.caas-1AL and TaLOX-B1 loci in Ningxia wheats were analyzed by using exsiting molecular markers.The results showed that the frequencies of different allelic variations and its combinations were different.At QLpx.caas-1AL locus,two new allelic variations were found and named Xwmc312-199 and Xwmc312-223,respectively,according to the length of their sequences.The frequencies of five allelic variations were 46.11%,32.78%,18.89%,1.11%,1.11%,respectively.There existenced two kinds of allelic variations at TaLOX-B1 locus,which were TaLOX-B1a and TaLOX-B1b,respectively,and which accounted for 15.56%and 84.44%,respectively.Eight kinds of allelic combinations were identified at locus of TaLOX genes,which Xwmc312-227/TaLOX-B1b?40.56%?shared the highest proportion,followed by Xwmc312-235/TaLOX-B1b?27.78%?.And the frequencies of Xwmc312-199/TaLOX-B1b?1.11%?and Xwmc312-223/TaLOX-B1b?1.11%?were the lowest.Meanwhile,differences of distributions of TaLOX genes among wheat cultivars and advanced lines from different regions and different sources in the same region were also found.Overall,the frequency of allelic variations with low activity LOX gene(Xwmc312-227/TaLOX-B1b)in Ningxia wheat cultivars and advanced lines was significantly higher than with high activity LOX gene(Xwmc312-235/TaLOX-B1a). |
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