The Construction Of Maize Phytosiderophore Secretion Characteristic Estimated System And Cloning Analysis About Iron Deficiency Related Gene Fragment

Iron (Fe) deficiency chlorosis (lime chlorosis) is a world widespread mineral nutrient deficiency in higher plants, especially corn, sorghum and fruit tree, grown on calcareous soils. Soil amendments and foliar sprays of Fe are not viable economically in the long term to correct Fe deficiency. Therefore, to alleviate Fe chlorosis in the long term, alternative methods, for example breeding of more chlorosis resistant genotypes (cultivars) are required. The recent hotspot in agricultural research is to reach the high yield, quality and efficiency target by the using of plant's stress tolerance. Graminaceous species can enhance iron (Fe) acquisition from sparingly soluble inorganic Fe (â…¢) compounds by release of phytosiderophores (PS) which mobilize Fe (â…¢) by chelating.Using corn inbred lines as material, this study had compared three PS amount test method, analized the PS secretion ability and iron sensitivity at seedling stage.While the different of PS secretion ability between seedling and heading stage and the regulation of PS secretion characteristic's inherit had been studied. Two SSH cDNA library which one was about the Fe deficiency metobolize regulating, another was about the different PS secretion ability had been constructed.Using the SSH cDNA library which one was about the Fe deficiency metobolize regulating, the cDNA which reduced by Fe deficency had been analized.The main results were summarized as follow:1 The Phytosiderophores (PS)'s test method had been set up.Using 16 corn inbred lines,according to the Fe(â…¢) chelated characteristic of mugineic acid, a new indirect method of inductively coupled plasma-atomic emission spectrometry (ICP-AES) and a modified direct method of ultra-spectra high performance liquid chromatography (UV-HPLC) for the measurement of MAs or deoxymugineic acid (DMA) were established in corn inbred lines. The comparison of these two methods with traditional spectrometry was also made. The results showed that the ICP-AES method, using Fe₂O₃ as the dissoluble object instead of the traditional Fe (OH)₃, was more stable and repeatalble suggesting that ICP-AES was a simple and quick method for evaluation of the MAs content in corn and other graminaceous crops. The modified UV-HPLC was also precise and efficient, and could be used for the direct mesurement of graminaceous's deoxymugineic acid. 2 The corn inbred lines PS secretion characteristic had been analized.The rates of phytosiderophore secretion had been tested by using ultra-spectra high performance liquid chromatography (UV-HPLC) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) with 46 corn inbred lines at seedling stage and heading stage. The iron deficiency sensitivity, the phytosiderophores secretion ability and change in different growth stage had been estimated. There was marked correlation existed in phytosiderophore secretion rate under Fe(+) and Fe(-) condition, and a marked correlation, r(Fe+) = 0.7149^**,existed in Fe(+) rates of phytosiderophore secretion between two test methods. The rates of phytosiderophore secretion of seedling stage and heading stage had marked correlation, r=0.9701^*. Between the phytosiderophores secretion rate in seedling stage and the decrease degree of phytosiderophores in heading stage, there was a marked logarithm correlation r=0.892^*.The corn inbred-lines could be clustered into 4 types with different phytosiderophores secretion rate and iron difficiency sensitivity using the median of Fe(+) phytosiderophores secretion rate [2.836 mg/(g^*per plant^*3h)] and the increment of phytosiderophores secretion rate after being Fe(-) treatment [0.209 mg/(g^*per plant^*3h)] as standard.The HPLC was an exact method in PS testing, but the ICP-AES could be used in material selection. The phytosiderophores secretion characteristic was different at different growth stage. An estimated system had been constructed using PS secretion ability and iron deficiency sensitivity, and corn inbred lines could been divided into four types. The type with low PS secretion rate and high iron difficiency sensitivity are the expected Fe-efficient nutrition breeding material.3. Genetic analysis of PS secretion characteristic8 inbred lines which has different PS secretion characteristic each were used to make 28 hybrids by (1/2) n(n-1) diallel crosses design.The PS secretion ability of the hybrids and parents had been analized. It had been realized that there was a highly significantly correlationship existed between the PS secretion ability and the GCA from same parent.The combination with top SCA maybe not the best PS secretion ability combination .Only when both combination SCA and parent GCA were high ,the combiantion with strong PS secretion ability could be got.The PS secretion characteristic was mainly controlled by dominant effect,and the additive effect was proved to be weaken.The genetic model of PS secretion abilitys were proved to match the additive-dominant model, and was controlled by at least 1 major gene. However the iron sesectivity was not,and it has ralative less major gene.The major gene group seemed not to be the same group, hence the dominant effect they controlled were opposition,which is possitive(iron sensitivity),another is negative(PS secretion ability).4 suppression substractive hybridizationUsing the root apex cDNA from Chang7-2 under normal treatment as driver and Chang7-2 under iron deficient treatment as tester, one suppression subrstractive hybridization had been handled. While another hybridizaiton the iron deficiency treatment root apex cDNA from Chang7-2 as driver and zheng 58 as tester had been done. The PCR product from specific expressed cDNA had been gotten. The secific expressed cDNA was associated with the control of iron deficiency and PS secretion ability.5 Analysis about the gene which controlled corn iron deficiency metabolizeSSH library had been constructed using the PCR product associated with iron dificiency. The lengh of segments which come from library were about 200 bp-900 bp.1230 clones had been choosen out and sequenced, 203 non-repeated sequence information had been got. Among them, there was 9 fragment could be putatived to be related with lower energy regulate after being iron deficiency treatmeng, 3 fragment was hypothetical iron oxidoreductase, 2 fragment may be associated with iron binding protein, 5 fragment may have the function about the membrane trasport,3 fragment was associated with aminotransferase, 5 could be putatived with plant resistant ability and 2 fragment were putative transcriptional regulator. 0257, a most special fragment within all fragment, was putative to has relationship with helix-turn-helix which was different to the bHLH (helix-loose-helix). The function of 0257 may be the negative control and had some touch with the negative dominateffec of genetic control. Besides, 77 fragments may be the new gene sequence, and the other was unknown function. Further reaseach should be done to explain the fragments's special expression with corn iron deficiency gene control.