The Analyse Of Transcription Factor ZmNAM056 Regulates Expression Of Genes Related To Starch Synthesis In Maize

Maize is the largest food crop in the world, the planting area and production ranks first in the three major grain crops, and corn starch is one of the best starch in the chemical composition. Now, with the world population increasing, resource and arable land area is decreasing, the crop yield and quality requirements are also getting higher and higher.The core of the biosynthesis process of starch includes synthesis the ADPG, extending the polysaccharide chain, branching polysaccharide chains and debranching the polysaccharide chains. starch Biosynthesis is a very complex process, completed by a series of enzyme catalyzing the synthesis of starch. There are four kinds of key enzymes involved in the final stage of starch synthesis in the endosperm:ADP-glucos pyrophosphorylase, Starch synthase, Starch branching enzyme, and Starch debranching enzyme. At present, the research of the function of the starch synthase has been relatively deep reasearched. But the research on the upstream regulatory mechanism of starch synthesis related enzymes, the research on the upstream regulatory mechanism of starch synthesis related enzymes, the enzymes associated with starch synthesis of cis-acting elements and transacting factors research reported little.According to the literatures and existing gene resources, we choice the genes which related to starch synthesis and express specifically or highly expressed in endosperm. this eight genes are the Sh2、Bt2、SS1、SSⅡa、SSⅢa、sbe1、 sbeⅡb、 Isa1. Our team has screened transcription factors can participate in synthesis the starch of corn in the endosperm by determinating the expression pattern of RNA in different key points during the grain filling periode, screening transcription factors which maybe involved in synthesis of starch in maize endosperm. At the same time, through the correlation analysis in public database, our research group has screened transcription factors which are related to above eight genes, choice the transcription factors which have high correlation coefficient as candidate transcription factors. By comparing the candidate transcription factors screened by the two methods, we found a common transcription factor of NAC family, and named it ZmNAM056. On the basis of this, we will use semi-quantitative RT-PCR or fluorescence quantitative PCR, gene expression, subcellular localization and yeast one hybrid to continue to validate the regulation of screened transcription factors for key enzymes of starch synthesis genes. The main results are as follows:1.Excellent maize inbred lines B73 as experimental materials, we has successfully cloned the transcription factor from maize endosperm, this gene has an open reading frame for encoding a protein of 391 amino acids. Homo logy to the amino acid sequence of other plans, funding the ZmNAM056 transcription factor contains the binding domain for DNA at the N terminal and a transcription activation domain at the C terminal.2.Analyzing the expression pattern of ZmNAM056. Semi-quantitative PCR results showed that the ZmNAM056 express specificity in seeds; Then the result of the real-time fluorescence quantitative PCR showed that the ZmNAM056 gene expresses is low in the first 7 days after pollination in the seed,10 days after pollination begin to express largely,20 days after pollination was the highest, and that of maize endosperm starch synthesis related genes showed a strong positive correlation. At the same time, the influence of different sugars and plant hormones on Endosperm starch synthesis related genes and gene expression of ZmNAM056 was also studied, and found AGPase gene Brittle-2 and Shrunken-2 in the treatment of its expression is consistent with the trend, induced by Glu, Suc, ABA, inhibition by Fru and Tre, starch synthesis enzymes (SSs)gene, SSIIa was induced by Glu, but not by ABA, SSIIIa was induced by ABA, but not by Glu, SSI was induced by Glu and ABA; The synthesis of starch branching enzyme (SBE) gene SBEI was induced by Tre and Suc, however SBEIIb were significantly induced by Fru and Glu. Results show that the same gene in different processing conditions, the expression is different. When different genes in the same processing conditions, the expression of some genes trend is consistent, but some expression trend is different. The expression of those genes are affected more by sucrose, glucose, ABA, but the expression of ZmNAM056 gene is not affected by the influence of sucrose; hormone ABA and glucose promotes the expression of ZmNAM056 gene. It is interesting, sucrose and ABA manage together, promoting effect is more obvious.3.Construct 35S::ZmNAM-GFP fusion protein expression vector, and conduct the subcellular localization by gene gun bombardment mediated transformation of onion epidermal cells, The results showed that ZmNAM056 protein located in the nucleus. The result of yeast transformation experiment shows that the ZmNAM056 gene encodes a protein has transcriptional activation. Cloning the coding sequence of ZmNAM056 protein in maize to insert into the pET-28 vector, in the strain of escherichia coli RosettaTM (DE3) to conduct prokaryotic expression. Then 6 X His-ZmNAM056 fusion protein was purified by Ni-Agarose column.4.By gene gun bombardment mediated transient expression experiments in maize endosperm and Yeast one hybrid experiment comfirmed the transcription factor ZmNAM056 binding to the starch synthesis related gene promoter, founding the expression of ZmNAM056 up-regulate Sh2 and Bt2 gene is likely to be through indirect effects of regulation, the upregulate of SSIIa and sbellb and inhibition of sbe1 gene transcription through direct and is binding to its promoter.