| Maize (Zea mays L.) is a major cereal crop for both livestock feed and human nutritionworldwide. Maize diversified uses as food, feed, industrial raw materials and so on, which has animportant strategic significance. In China maize has the highest growing area. But average yield is notas high as other top yielding countries. Therefore, the main task is to improve maize yield, especiallyyield per unit area. However, like other cereals normal maize contained low quality proteins and lackedessential amino acid, such as lysine and tryptophan, wich restrict the nutrition value of maize. So thequality improvement of maize needs an important breeding goal. The opaque-2(o2) mutant gene canlargely enhance the essential amino acid content, such as lysine and tryptophan, in maize. Moreover, themutant gene has been investigated thoroughly, and can be used in production. A good example is thesuccessful useage of quality protein maize (QPM), which has hard or semi-hard endosperm andmaintains the content of lysine more than twice that of normal maize, which maize its superior proteinquality and protein digestibility over normal maize. While most of the QPM germplasms are tropic orsubtropic lines that do not fit for the direct cultivation in temperate areas. Back cross breeding canintrogress the o2mutant gene into receptor lines, which can be combined with marker-assisted selectionto introgress the o2mutant gene of QPM into normal maize inbred lines can be suited for temperateareas cultivation. In this way, we can get many o2near-isogenic lines, can be constructed and enlargethe germplasms of high lysine maize. However o2gene has pleiotropic effects, the increase extent oflysine content exists difference in different genetic backgrounds, such as the discovery of o2non-highlysine maize. So a easy and effective way of monitor the content of lysine is needed in the process ofbreeding. In our research, we introgress the o2mutant gene of two different kinds of QPM into the localnormal inbred lines, and constructed many o2near-isogenic lines. Then using our improved dye bindinglysine (DBL) method to monitor the lysine content, which can provide a reference for construction ofhigh lysine maize germplasms. Besides these, our research also analyse a group of specific o2near-isogenic lines to investigate the reason why they exist distinct kernel phenotype, and analyse withproteomics to discuss the function of o2mutant gene. Main research content are as follows:1. We selected two kinds of QPM inbred lines CA339and Shandong2548as donors, some Chinesewaxy and normal maize inbred lines as receptors. Then analyse the polymorphisms of donors andreceptors, using the simple sequence repeat marker phi057to confirm the hybridized combination andgenotype, at last we construct the o2near-isogenic lines. The results indicated that, polymorphisms notonly exist in donors, but also exist in receptors. In order to successfully construct the o2near-isogeniclines we must choose the correct donors and receptors for hybridized combination. Only in this way wecan use marker-assisted selection to select the correct genotype, and we find and demonstrated thatdonor Sandong2548can be used to construct o2near-isogenic lines.2. According to the previous researches and national standard GB4801-84, we improved the DBLmethod. In this paper, we have measured the lysine content of18groups of successfully constructed o2near-isogenic lines. The results demonstrate that, lysine content among normal inbred lines varied from 0.223%to0.368%, on an average of0.317%. On the other hand, among o2near-isogenic lines it variedfrom0.373%to0.527%, on an average of0.430%. Compared with their own receptors, lysine contentimprovement varied from13%to74%. Among the four groups of o2near-isogenic lines constructed byShandong2548, average lysine content is0.440%, while the other fourteen groups of o2near-isogeniclines constructed by CA339showed0.428%, their corresponding receptors are0.315%and0.317%,respectively. By contrast, the donor Shandong2548had increased tendency of lysine content was CA339,but has no significant difference among these donors.3. We discussed the reason why a group of specific o2near-isogenic lines existed distinct kernelphenotype. Therefore, we analyse the phenotypes, genetic similarity, gene sequences and structure, geneexpression quantity and so on. The results indicate that lysine content of liao2345/o2-1, liao2345/o2-2,and liao2345are0.32%,0.49%, and0.28%, and endosperm phenotype are hard vitreous, floury opaque,and hard vitreous respectively. Plant phenotype indicates that compared with liao2345, liao2345/o2-2ismore resemble to liao2345than liao2345/o2-1. The genetic similarity analysis had shown that comparedwith liao2345, the similarity of liao2345/o2-1and liao2345/o2-2are0.85and0.91, respectively.Mutiple alignment of the CDS demonstrated that both of the two o2near-isogenic lines acquired thesequence of CA339, what make the difference is that liao2345/o2-1has wild type promoter, whileliao2345/o2-2has a insertion of rbg transposable element in the promoter. RT-PCR analysis indicatedthat the expression quantity of liao2345/o2-1is equal to liao2345, they have the normal expressionquantity, while the expression quantity of liao2345/o2-2is largely inhibited. So we like to conclude that,the OPAQUE2protein of CA339owns normal transcriptional activity, and its mutation belong to nullmutation. Two o2near-isogenic lines possessed different promoter sequences owing to differentcrossing over or recombination mechanism, one has the wild type promoter of liao2345and the otherhas the mutant promoter of CA339, it is the reason why they have instinct kernel phenotype.4. According to the protein two dimensional gel electrophoresis and mass spectra analysistechonogy, we analysed the protein composition of the18DAP endosperm of liao2345/o2-1,liao2345/o2-2, and liao2345. The results demonstrated that owing to the different function at o2locus,the protein composition has a big difference and there exists many differentially expressed proteins,which may under the control of o2locus. We also identified25proteins, after analysis, we confirmedthat they are mainly some kind of enzymes, and they are involved in different metabolic processes in theorganism, such as sugar and protein metabolism. |
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