Study On Quality Related Genes Avenin-like B And NAM-B1 Of The Wheat And Related Species

Wheat is one of the major food crops in the world.With the changing of market demand,people pay more and more attention to the quality of the wheat.Wheat quality is determined by the combination effects of many factors such as protein,lipid and starch,among which the protein content,quality,and interaction are especially important.The seed storage proteins consisted of glutenin and gliadin in wheat,which positive correlated closely to dough rheological properties.The glutenins are the major components of wheat storage proteins,which consist of high-molecular-weight glutenin subunits(HMW-GS)and low-molecular-weight glutenin subunits(LMW-GS).Gliadin including ?-? ?-? ?-and?-gliadin.However,30%-70% of the wheat quality variation can be explained in the traditional wheat gluten.Although cysteine content is very low in amino acids of traditional gluten protein,the effect of cysteine residues can be to make the protein structure into polymers through intermolecular disulfide bond.A new found storage protein,avenin-like,is caught a great attention for its number of cysteine residues in recent years.It is apt to form inter-disulfide bonds or intra-disulfide bonds with neighboring storage proteins,which may broaden the protein-nets and develop elasticity in a dough,resulted changing the processing quality.In fact,research in this field has just been started not long ago.There still remained a paucity of genetic information about the chromosomal location,number of loci and alleles,and allelic effects.The ancestral NAM-B1 allele has been discovered in many tetraploid wild emmer(T.turgidum ssp.dicoccoides)accessions and contributed to the rate of senescence and allowed more effective translocation of nutrients to the grain,resulting in shorter grain filling and higher concentrations of protein,Fe,Zn,and Mn in the grain but reduced yield.To date,there are few studies on the distribution of the ancestral NAM-B1 allele in Chinese wheat cultivars.The goal of this study was to identify the NAM-B1 alleles(wild-type,1-bp insertion and deletion)in Chinese cultivars from different geographical areas and provide some information for improving wheat quality in China and other countries.The genes of avenin-like b and NAM-B1 are the primary focus in our study due to affecting the protein quality and content,respectively.The major results of this study are listed as the following:1.Using two-demensional electrophoresis(2-DE)method,we analyzed the proteinsprofiles of L16-3BS which were chromosome segment substitution lines.50 differentially expressed protein spots with higher than 2-fold spot thicknesses were found in gel image.These protein spots were identified with MALDI-TOF/TOF and 47 spots were identified successfully.Compared to control,the abundance of 35 proteins increased and of 10 decreased.As follow,Globulin(5),Alpha-gliadin(2),gamma gliadin(2),MYB transcription(1),elongation factor(1),Triosephosphate isomeras(2),Serpin(7),malate dehydrogenase(1),avenin-like(2),triticin(3),Superoxide dismutase(1),Heat shock protein(2)and unknown protein(18).Avenin-like b genes was choosed as candidate genes to study.2.The avenin-like genes were cloned by gene specific primers,followed by sequencing and bioinformatic anayzing.The coding gene sequences were 855-858 bp long and no introns.Chinese Spring nulli-tetrasomic confirmed that type-b ALP genes of wheat belong to a multigenefamily.Three genes of type-b ALPs were identified and mapped to chromosomes7AS(TaALPb-7A),4AL(TaALPb-4A)and 7DS(TaALPb-7D).Further comparison revealed that the TaALPb-7A gene had three alleles,designated TaALPb-7A1,TaALPb-7A2 and TaALPb-7A3.While TaALPb-4A gene had four alleles,TaALPb-4A1,TaALPb-4A2,TaALPb-4A3 and TaALPb-4A4.The TaALPb-7D locus did not show any allelic variation.The phylogenetic relationship of the 42 cloned type b ALPs sequences was analyzed by applying UPGMA to the aligned complete coding sequences of all clones and wheat storage protein genes,as well as the reported ALPs of wheat-related species available from various databases.The cloned type-b ALP sequences clustered according to their chromosomal origin.The cloned ALP sequences were closest to the reported type-b ALP sequences of related species,followed by sequences corresponding to HMW-GS and LMW-GS,while ?-gliadin were the furthest in evolutionary terms.3.Results of ddPCR revealed that the cultivars with the active allele give a normal gene expression,the ratios of expressed gene copy numbers between TaALPb-7A and actin ranged from 1:2.54 to 1: 3.36,while the four cultivars with the silent allele had no gene expression.4.TaALPb-7A1,TaALPb-7A2 were cloned into the expression vector,and transformed into host bacteria Escherichia coli Rosetta-gami BL21(DE3).After induced by IPTG,the fusion protein was purified and then been integrated into the control flour to determine the quality effect utilization of Mixograph.The results revealed that TaALPb-7A1,TaALPb-7A2 has an important effect on processing quality.5.The fact that the TaALPb-7A locus has two types of alleles,active and silent,allowed us to study its allelic effects.Allele-specific PCR markers were designed to differentiate the two types of TaALPb-7A alleles.Significant allelic effect differences were detected bewteen the active and silent alleles of TaALPb-7A.The active allele was significantly associated withhigher dough strength parameters,including Midline Peak Time(P<0.0443),and Midline Time x Width(P<0.0096).No significant association was detected between the allelic types and grain protein content or gluten content,indicating the high dough strength of the active allelic type is from the expression of TaALPb-7A.6.The avenin-like genes were cloned by gene specific primers,followed by sequencing and bioinformatics analyzing in 12 genomes(W?E^e?E^b?St?Q?Ns?Ta?U?C?N?V ? R)of the wheat related species.Results suggested that abundant genetic variation and mutation of avenin-like genes were found in some genomes,which is available for incorporation into cultivated wheat by wide crossing.7.This study investigated the NAM-B1 alleles in 218 Chinese cultivars that were collected from five major wheat regions(12 provinces),covering most of the winter wheat growing regions in China.The results showed that the NAM-B1 gene is present in 53(24.3%)cultivars.The remaining 165(75.7%)cultivars do not contain this gene.Thus,exploring the distribution of NAM-B1 allelic variations(1-bp insertion and deletion)can provide some information for improving the quality of winter wheat in China and other countries.