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Cloning and Sequence Analysis of Genes and Promoters of ZmNAS1 and ZmNAS2 in Different Maize Inbred Lines

Author: LiJun
Tutor: ZhangChunQing
School: Shandong Agricultural University
Course: Crop Genetics and Breeding
Keywords: Maize ZmNAS1 ZmNAS2 Coding region Promoter Clone elements analysis
CLC: S513
Type: Master's thesis
Year: 2011
Downloads: 63
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Abstract

Iron is one of necessary nutrient elements in plant growth process. Iron element is existed by not soluble and difficult to be absorbed form in calcareous soils. Therefore, iron deficiency is more prevalent. Graminaceous species can enhance iron(Fe) acquisition from sparingly soluble inorganic Fe(Ⅲ)compounds by release of phytosiderophores(PS) which mobilize Fe(Ⅲ) by chelating, such as corn. ZmNAS is a key enzyme in phytosiderophore synthetic pathway in maize. The main results of this study as follows:1. 24 inbred lines’ ZmNAS1 and ZmNAS2 sequence of coding area were cloned, The consistency of ZmNAS1 coding area DNA sequence is 99.43%. There were 38 variable sites, accounted for about 3.9% of total sites and a variable site every 26 bp sites on average. In which single information site is 16, and parsimony-informative site is 22. There is no insertion and deletion. The numember of amino acids synonymous mutations are 17 and missense mutations are 21. The PI of ZmNAS1 coding areas nucleotide diversity is 0.00884, and the HD(haplotype diversity index) is 0.938±0.00155. 17 kinds of haplotype are defined by 24 sequence. The consistency of ZmNAS2 DNA encoding region sequence among the 24 materials can reach up to 99.16%; and the variable sites were founded 60 in all, Average 30 bp appear a variable site. In these variable sites, 9 single variable sites, 51 parsimony-informative sites, no insert and deletion. 35 amino acids synonymous mutations, 25 missense mutations. The PI value of the nucleotide diversity of the encoding region of ZmNAS2 gene is 0.01191 and the HD(haplotype diversity index) is 0.964±0.00057. 17 kinds of haplotype are defined by 24 sequence. Compared with previous studies, in general, single nucleotide mutation rate of two ZmNAS coding region is higher. ZmNAS1 encoding region have more low-frequency SNP than ZmNAS2.2. ZmNAS1gene promoter sequence were cloned in 24 maize inbred lines, According to the sequence of the promoter of the ZmNAS1 gene of 24 materials, analyzed the gene polymorphism among the different maize inbred lines. The similarity of the promoter region reach 92.05%, the longest is 2315 bp and the shortest is 1741 bp. There are 5 single information sites, 65 parsimony-informative sites, deletion and insertion 587 bp. The value of PI is 0.01359. 13 kinds of haplotype is defined. HD (haplotype diversity index) is 0.917. Compared with coding region, the promoter mutation were more active.3. The test of natural evolution by Tajima’s D value、Fu and Li’s D* and F* values,results show that coding regions and promoter of ZmNAS1 and coding regions of ZmNAS2 were not deviating from the neutral evolution ,It shows that they less affected by natural selection.4. Use bioinformatics software to analyze ZmNAS1 and ZmNAS2 gene promoter elements and compare the differences among materials. The results show that ZmNAS1 and ZmNAS2 gene promoter have some cis-acting elements related to light regulation, hormone induction, response stresses and so on. But there is some different number. In addition, there are a certain number core sequence of the iron-deficiency induction elements on the two gene promoter of different materials. According to promoter elements analysis, we pre-judge that ZmNAS1 and ZmNAS2 gene expression is a complex control process. It may be controlled by many other signals besides the iron deficiency. We speculate that regulating level on expression is also different in Various materials according to the difference of gene promoter.5. Homology cloning on the ZmNAS1 genetic encoding area of the rice, sorghum, barley, wheat, rye, oats and hullessbarley by the primer of cloned gene of ZmNAS1 encoding region in maize inbred line. Including corn ,The consistency of encoding region sequence of NAS1 gene among the eight species can reach up to 99.58%, It is even exceed the consistency of 24 maize inbred lines. It shows that the gene of NAS1 is highly conversed in the species evolution. The variation site also appears in the different maize inbred line. It shows that the variation sites have been existed before the species differentiation.

Cloning and Sequence Analysis of Genes and Promoters of ZmNAS1 and ZmNAS2 in Different Maize Inbred Lines

Abstract

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