| Today, over three billion people are afflicted with micronutrient malnutrition and the numbers are increasing. Developing micronutrient-enriched staple plant foods, either through traditional plant breeding methods or via molecular biological techniques, is a powerful tool to combat micronutrient malnutrition. As the second largest crop, wheat contains insufficient amounts of many essential vitamins and minerals to unmeet the human needs just as other major staple crops. So the significance to determine and analyze the micronutrient content in different varieties of wheat, study the genetic mechanism of micronutrients and clone important micronutrient-related genes in wheat is self-evident.The contents of micronutrients including V_C, antioxidant substance (SOD, GSH, GST, GSHP, GR, TAS), major and minor elements ( K, Ca, Mg,Na, Cu, Fe, Zn and Mn) in 286 varieties of wheat seed, were determined and analyzed. Thousand-grain-weight, sprout rate and grain polymorphism were also studied. The data distributions of 14 micronutrient contents can be divided into three groups, the data distribution of GSHP, GSH content belongs to normal distribution; the quasi-normal distribution included the content of GST, Vc, SOD, TAS, K, Zn, Na; the third group is non-normal distribution, the distribution of Ca, Cu, Fe, Mg and Mn belongs to this group. The characters owned normal distribution are considered as quantitative characters and the QTLs of these characters would be detected easily. The content variation of trace element is at the range of 3.20-1.43 multiple. The difference of Na content in 286 varieties of wheat seeds is 3.20 times which is the largest. The variation multiple of Zn and Cu content is 2.23 and 2.13, respectively, which is the second largest. The content of Mn varies least whose variation multiple is 1.43. The content variation of anti-oxidant substance is at the range of 13.97-1.34. The difference of Vc is 13.97 times which is the largest. The variation factor of GST and GSHP content is 9.12 and 6.13, respectively, which is the next largest. The content of SOD varies least whose difference is 1.34. Some materials, such as Damuzhiai, Pml6, Pml9, Pingyang 181 and Nanda 2419 etc. which owned two or more than two maximum or minimum value, are important to breeding and genetic mechanism research. Some negative correlation exists between characters of wheat seeds and micronutrient content. Major correlation lies among different anti-oxidant substances and trace elements respectively, but little correlation was found between antioxidant substances and trace elements. These phenomena remind us there are two sorts of mechanism for the wheat seeds to store antioxidant substance and trace elements respectively, and little cross exists on the two sorts of mechanism. Some materials owned similar genetic background were clustered based on micronutrient contents and characters of seeds which reveal that the genetic factor is major even though the environmental factor couldn't be neglected.A set of 114 recombinant inbred lines of the 'International Tricia Mapping Initiative' mapping population was grown during the seasons 2002 and 2003 under different wheat regions in China. Twenty- two traits including antioxidant substances (Vc, V_E, SOD, GSH, GST, GSHP,GR, TAS) ,macro and micro elements ( K, Ca, Mg, Na, Cu, Fe, Zn and Mn) , Amylose, total starch content in wheat seeds, thousand-grain-weight and grain polymorphism ( grain's color, size and fullness ) were researched. The genetic map with 846 markers was constructed through the MAPKER 3.0 using the separate data of 938 markers published in the GrainGenes database. QTLs with additive and espistatic(esp.) effects were mapped based on mixed linear model approaches by QTLMapper version 1.0. 18 additive and 15 pairs of espistatic QTLs related to micronutrient content of wheat seeds were detected and should be considered as new QTLs for no reference-retrieved reports about QTLs of micronutrient content in wheat. Some overlaps or strict linkages were observed through viewing over the distribution of all additive and espistatic QTLs. First and most interesting overlap was on the interval of Xfbb377-Xfbcd410 at 2DL distal. One esp. QTLs of the SOD content in wheat seeds and one additive QTLs of total antioxidant status of wheat seeds located in this interval. As we all known, SOD is one of the important antioxidant substances. So the conclusion can be drawn that the region contains some genes related to SOD content in wheat seeds considered the result of Neuman P. R. and Hart G E.(1986) . Another overlap should be mentioned that one esp. QTLs of VE and one additive QTLs about Vc content in wheat seeds located at the interval of Xpsr463-Xpsr915 on 6AL. The locations of two QTLs of Vc ( add., esp. each) and one additive QTLs of VE on the 2BS distal are coincident linkage strictly. Both Vitamin C and Vitamin E are important and necessary micronutrient for human being which are related to antioxidant activity. Two reasons maybe explain this overlap and linkage. Firstly, the storage mechanisms of Ve and Vc in wheat seeds share some same ways; secondly, part genes related with the VE and Vc content are linkage strictly. Sixty-six additive and espistatic QTLs related to 19 traits distributed on 19 chromosomes except for 1A and ID (Table 3), but the distribution is uneven. Six esp. and three add. QTLs were located on 2B chromosome which owned the most QTLs. Only one esp. QTLs located on 7B. Among the seven homologous groups, the second group has the most QTLs where situated at 14 QTLs. 2 QTLs lied on the first group which owned the least QTLs. Twenty-eight add. and esp. QTLs situated on group B chromosomes which owned the most QTLs among groups A, B, and D. Sixteen QTLs were identified on group D which owned the least QTLs. This result provides a reference for the sequencing project of gene rich region of wheat.Ferritin is a large multi-subunit protein that stores iron in plants, animals, and bacteria. Both cDNA and gDNA of Ferritin from wheat based on homologue sequence have been firstly cloned. The ferritin genes are organized with eight exons and seven introns. It is proved a single copy in the genome of wheat through Southern blot test. The expression of the gene varies in different tissues (green seeds>root >leaf) . The SSR primers were designed based on the repeat sequence (CCG) near the starter codon. The polymorphous amplification of the PCR product through the SSR primers was observed among 286 varieties of wheat. The amplification fragment with least Mw is negative correlation with the content of iron remarkably. The polymorphous amplification is caused for the difference of the repeat sequence unit through DNA sequencing. Alanine chain near N-end of the peptide sequence appears 1-2 Alanine absence, which would affect the iron storage in wheat seed.Both cDNA and gDNA of GST gene have been cloned firstly from wheat by homologue sequence. The GST genes are composed of three exons and two introns. It belongs to a single- or oligo- copy in the genome of wheat by Southern blot test. The expression of the gene enhances obviously in different tissues and growth stage. There are two kinds of GST sequence in Chinese Spring. Comparison of two sequences shows an identity of 95%. The major difference appears on introns. The first kind sequence is absent of 13bp and 7bp on the two intron regions, respectively, which leads to the first kind sequence (1054bp) (CSS-1) is shorter than the second kind of sequence (1084bp) (CS S-2) . Only one kind of cDNA sequence was detected in green seeds, which is the expressed product of the first kind of sequence. Only the first kind of sequence was detected in other varieties, such as Opta85, W7984, Shanhongmai and Wenmai 6#. Three kinds of GST sequence were detected in the relative of wheat: the first kind of sequence has two introns and identifies to CS S-l; there is no intron in the second kind of sequence which are homologous to cDNA sequence; the third kind also have two introns, they are homologous to CS S-2. The primers were designed based on the flank sequence of introns. There's no polymorphous amplification of the PCR product from the primers designed on the basis of the conservative sequence of the first and third exon of GST gene among 286 varieties of wheat, but the polymorphous amplification among the materials of genome A, S, D in diploid species of Triticeae was observed. The different genomic materials could be distinguished through the molecular weight of the amplification fragment. The polymorphous amplification is caused by the absence of 4bp, 13bp and 9bp in the intron regions among the materials of genome A, S, D in diploid species of Triticeae by sequencing. The largest amplification fragment (263bp) is specified with genome D materials, the second largest amplification fragment (259bp) is specified with genome S materials, the amplification fragment of 250bp belongs to the genome A and the fourth (241bp) is common to all materials. The GST gene is located on chromosome 3 A, 3B and 3D of wheat through using nulli-tetrasomic lines of Chinese Spring.Both cDNA and gDNA of Zip gene, which is related to the zinc absorption and tansportation from wheat with homologue sequence, were also firstly cloned. There is no intron in Zip gene. It is proved a single copy in the genome of wheat with Southern Blot test. The expression of the gene varies little in different tissues (green seeds, root, leaf) and growth stage. There are two kinds of Zip sequence in Chinese Spring. Comparison of two sequences shows an identity of 97%. There are 26 base substitutions between two kinds of Zip sequence, which lead to 8 amino acids' difference, and the different amino acids are not located in the conservative domains of Zip genes. Two kinds of cDNA sequence were detected in green seeds, which is the expressed product of the two kinds of genomic sequence. 15 of 21 varieties with different zinc content owned two kinds of Zip sequence which is identified with two kinds of sequence from Chinese Spring, respectively. There is absence or insertion of single base on the Zip sequence of 6 out of 11 varieties with low zinc content, but no absence or insertion of single base on the sequence of 10 varieties with middle or high zinc content. Any absenceor insertion of single base leads the malfunction of the translated protein because the Zip sequence has no intron. The SSR primers were designed based on flank repeat sequence of repeat unit near the start codon. No polymorphous amplification of the PCR product through the SSR primers was observed among 286 varieties of wheat, and neither among 29 relatives of wheat.The amylose and total starch content are important guidelines which determine wheat quality. They are also immediately associated with the health function of wheat. Quick, simple and minimal-quantity method which determines amylose and total starch contents is important to both selecting and breeding of excellent quality wheat and processing research. A novel micro-amount method with spectrophotometer(MAS) to determine amylose and total starch content of wheat seeds was discovered on the basis of national standard GB7648-87 by leaving out degreasing, decreasing the temperature, increasing time of dissolution in base and reducing the amount of sample. Determining Results of amylose content showed that parallel errors of 10 wheat samples using the MAS were less than 2% by lOmg sample with non-degreasing and basal dissolution at 65°C for 12hr; the errors between MAS and GB7648-87 method were also less than 2%; both kinds of errors are accepted according to GB7648-87. The total starch content was determined at the same time using the standard curve of water-solvable starch, the errors of two parallel experiments in group and between groups of MAS and GB are less than 1%, which are also permitted according to GB. The researched results indicated that MAS is characterized with a little amount of sample (1 /10 of GB) , no need of degreasing, lower temperature, lower pollution, time and labor saving, quick and acuteness, quality-repetition, and easy batch determination.In general, The content of micronutrients including Vc, antioxidant substance (SOD, GSH, GST, GSHP, GR, TAS), major and minor elements ( K, Ca, Mg, Na, Cu, Fe, Zn and Mn) in 286 varieties of wheat seed were determined and analyzed. QTLs with additive and espistatic (esp.) effects were mapped based on mixed linear model approaches by QTLMapper version 1.0. 18 additive and 15 pairs of espistatic QTLs related to micronutrient content of wheat seeds were detected and should be considered as new QTLs for no reference-retrieved reports about QTLs of micronutrient content in wheat. We have firstly cloned both cDNA and gDNA of Ferritin from wheat based on homologue sequence. The ferritin genes are organized with eight exons and seven introns. It is proved a single copy in the genome of wheat with Southern blot test. The expression of the gene varies in different tissues (green seeds, root, leaf). Both cDNA and gDNA of GST gene have been cloned firstly from wheat by homologue sequence. The GST genes are composed of three exons and two introns. It belongs to a single- or oligo- copy in the genome of wheat by Southern blot test. The expression of the gene enhances obviously in different tissues and growth stage. Both cDNA and gDNA of Zip gene, which is related to the zinc absorption and tansportation from wheat with homologue sequence, were also firstly cloned. There is no intron in Zip gene. It is proved a single copy in the genome of wheat through Southern Blot test. The expression of the gene varies little in different tissues (green seeds, root, leaf)...
|
PDF Full Text Request