| In recent years, with the rapid development of molecular biology and genomics, much more progresses in the study of characters of high yield and the mechanisms for high yielding, and their related genes have been made. Applying genetic engineering technology to the crop genetic improvement has become an effective way for increasing crop yield. Grain production had been significantly increased by using the major semidwarfing genes. Main problems existing in the crop breeding was simplification of genetic background and dwarf genes resource, because just only the dwarf genes Rhtl, Rht2, Rht8, Rht9 and sd-lwere predominant dwarfing gene present in current wheat and rice cultivars. A tiny minority dwarf resouces capable of being used in crop breeding, due to some dwarf resources accompanied by bad agriculture character. Therefore discovery and identify of new dwarf resources should be extremely important, the study and use of the dwarf genes are also taken more seriously.A spontaneous dwarf mutant was found in Dasypyrum villosum populations in 2000. The main purpose of this study was to study the genetics and morphological character, the physiologic effects by exogenoously application hormones on the dwarf mutant, the difference of gene expression level, which encoding cirtical enzymes in GA biosynthesis between wild type and dwarf mutant plants of D. villosum. In addition, GA3ox and GA20ox are cloned and its expression pattern is researched.1. We compared the morphological character between the wild type plants and dwarf mutant. The dwarf mutant showed no difference with wild type plants in seedling stage. The difference of height between wild type and mutants appeared from jointing stage, and the difference was up to the max until mature stage. At mature stage, the average height of wild type plants were 119.1cm and the dwarf plants were 33.1cm. The height of the mutant plant was only one fourth of the normal plants due to the shortened internodes and the decreased internodes. The dwarf mutant also showed partly male sterile. Pollen fertility test indicates that almost all pollen (95%) of the mutant is sterile, but only 10% pollen of the wild type plant were sterile. 2. In order to study the genetic of the dwarf trait, dwarf trait was crossed with the wild type plant, and the F1, F2 and BC1 populations were used for the genetic analysis of the dwarf trait. The plants of the F1 showed a wild type phenotype. The F2 populations exhibited a wide range of variation for plant height, and the F2 generation was segregated into two phenotypes:wild types and dwarf mutants. Based on the X2 test, the two phenotypes segregation reatios of the F2 population conformed to the ratio of 3:1. The BC1 population ((wild type X dwarf mutant) X dwarf mutant) showed two phenotypes:wild type and dwarf mutant with the expceted ratio of 1 wild types:1 dwarf mutants. There results confirmed that this dwarf phenotype is controlled by a single recessive gene.3. To assay the response of dwarf mutants to exogenous hormones, GA3, dwarf mutants were sprayed with 5 different concentration levels of GA3 (10"3-10-7M) the main results were as followed:the plant height of dwarf mutant can be rescued by the application GA3 continuously in different degrees. The higher the GA3 concentration was, the better the recovery effect was, in certain range. These indicated the dwarf mutants were sensitive to GA3, further study results showed that the recovery of plant height in dwarf mutants was resulted from the increasing of stem length, and 10-4 M GA3 is the best concentration level to spray with dwarf mutants. In additon, pollen fertility could be returned to wild tpye level afer spraying with GA3. Therefore, we infer that the dwarf mutant respond to GA application and is a GA-sensitive dwarf mutant.4. Based on the known GA 20-oxidase (GA20ox) cDNAs of barley and wheat, oligonucleotide primers were designed to isolate GA20ox genes from genomic DNA of D. villosum. Three haplotypes were obtained from the wild type and dwarf mutant,1293bp, 1297bp and 1294bp in length, respectively, in which haplotypeâ… andâ…¡were found in wild type and haplotypeâ…¡andâ…¢were found in dwarf mutant. Twenty-one SNPs and 4 InDels were found and could separate haplotypeâ… fromâ…¡. In coding region, eight SNPs lead to seven amino acid variations.The polymorphic sites in exonl and intron of GA20ox haplotypeâ…¢except positions 507 and 578 are the same as the haplotypeâ…¡, but the rest polymorphic sites in exon2 are equal to the haplotypeâ… . There was a C at position 507 in haplotypeâ…¢, whereas G and A was on the same site in haplotypeâ… andâ…¡, respectively. There was a T in both haplotypes in dwarf mutant, while InDel or T was on the same position in wild type. It is postulated that haplotypeâ… andâ…¡were the completely different haplotypes or haplotypeâ…¢was the variation of the haplotypeâ… because of the similar expression trend during the development of the plant.Q-PCR analyses showed that GA20ox three haplotypes were expressed in vegetative and reproductive organs. The total expression levels of GA20ox presented downtrend in leaf blade and ascend in stem, eustipes and developing spike along with the development of plants, respectively. However, they were firstly increased and then decreased in root from seeding stage to heading stage. These results revealed that the genes expression profile of GA20ox wasclosely related to the growth and development of D. villosum. Further analyses of the expression patterns of haplotypeâ… andâ…¡,â…¡andâ…¢were not significantly different in all tested tissues. It is postulated that they took effect simultaneously and haplotypeâ… /â…¢might have a synergistic effect with haplotypeâ…¡in GA biosynthesis, might even be able to substitute each other to a certain degree.The expression level of haplotypeâ…¡at all tissue and stages except the leaf blade at jointing and heading stages in wild type were higher than that in dwarf mutant. And the expression level of haplotypeâ…¢at all tissues at three stages except the leaf blade at jointing and heading stages and root in seeding stage in dwarf mutant were lower than haplotypeâ… in wild type. It means that tissues with higher GA20ox transcriptional level have more precursors to synthase more active GAs.5. Based on the known GA 3-oxidase (GA3ox) cDNAs of barley and wheat, oligonucleotide primers were designed to isolate GA3ox genes from genomic DNA of D. villosum. Two haplotypes were obtained from the wild type and dwarf mutant,1495bp and 1485bp in length, respectively, in which haplotypeâ… was found in wild type and haplotypeâ…¡was found in dwarf mutant. Twenty SNPs and twenty-two InDels were found and could divide haplotypeâ… fromâ…¡. In coding region, only two SNPs were non-synonymous mutation which led to two amino acid changes.Q-PCR analyses showed that both haplotypesâ… andâ…¡were expressed in vegetative and reproductive organs. Similar expression levels were found haplotypesâ… andâ…¡in three stages. The common features are generalized based on the expression level of GA20ox and GA3ox.These results revealed that the genes expression profile of haplotype I and II were closely related to the growth and development of D. villosum.In jointing stage, the transcript levels of both GA20ox haplotypes in leaf blade and developing spike of dwarf mutant are higher than that in wild type, whereas, the reverse happened in GA3ox. In addition, the expression level of GA20ox and GA3ox in root, stem and eustipes in wild type were higher than that in dwarf mutant imply that more active GAs were synthesized in those tissues of wild type. Maybe it is the molecular basis of phenotypes difference because plant height increased the fastest in this period, in which appeared the plant height differcne.6. Based on theQ-PCR results, CPS, KS, KO, KAO and GA2ox genes have differential expression patterns in all tested tissues, including leaf blade, root, stem, eustipes and developing spike.The expression pattern of the five genes were of spatial-temporal expression speciality during the plant development. The expression of CPS was no obvious difference between in tested tissues. The expression of KS was high in stem and eustipes, but low in root and leaf blade. KO was expressed highly in root, followed by eustipes. KAO and GA2ox were highly expressed in leaf blade and root, next to stem, eustipes and developing spike.The genes expression level of CPS, KS, KO, KAO, GA20ox and GA3ox in leaf blade of wild type were higher than that in dwarf mutant at seeding stage, contrary results found in root. It is possible that root was stimulated to synthesize more active GA to supply the growth demand by the homeostatic mechanism, and there was no phenotype difference.On the whole, the expression levels of CPS, KS, KO, KAO, GA20ox, GA3ox and GA2ox in leaf blade, root, stem and eustipes of wild type were higher than that in dwarf mutant at jointing stage, especially in root. And the highest expression level of these genes were in root at that time.We concluded that the stem, eustipes and root were the GA synthesis sites, and root is the most synthesization site of GA, part active GAs or GA precursors were transported from root or other organs to stem and eustiopes for the demand of plant gorwth.The expression patterns of CPS, KS, KO, KAO and GA20ox genes were similar, and the expression level of KS of wild type was no obvious difference with dwarf mutant. However, the expression level of GA3ox of wild type were mostly higher than that in dwarf mutant at the same organs and stages, even at the organs in wild type which the expression level of GA20ox were lower than that in dwarf mutant. GA3ox gene is the last important enzyme to control the active GA synthesis. Therefore, we populated that the abnormal expression of the GA3ox led the dwarfing.
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