Identification Of Endosperm-specific Expression Geens Of EXPANSIN Gene Family In Maize And Functional Analysis Of ZmEXPB13

As one of the most important food crops, maize serves not only as food for human beings and animals, but also as important raw material for industrial production. One goal for breeders and molecular biologists is to breed high-yield and high-quality maize. To improve the seed size is considered crucial to increase maize yield, which is decided jointly by the size and number of the cell, therefore, an effective way to improve maize production is to increase the cell size. Previous studies showed that activiating the loosening of cell wall is quite significant in regulating and controlling the cell size. Nowadays, researches focus on applying the technology of excessive expression to genetic improvement of crops. Since endosperm is an important component of seed, breeders and molecular biologists try hard to make clear the development of its cells, which is important to breed high-yield and high-quality maize, therefore, to discover the important functional gene which influence the size of endosperm cell of maize is an effective way to increase maize production. By employing the means of bioinformatics, this study did gene identification of expansin famiy of maize, make analysis of its structural features, evolutionary relationship, etc, and by studying the expression patterns of these genes, discovering the specific expression gene of endosperm, and meanwhile studing the influence of ZmEXPB13 on seed size by way of over-expression, and make preliminary research on the mechanism of action of expansin gene, the author has some conclusions as follows:1. By Blast homology search, we identified 88 expansin protein sequences from the genome database of B73, and divided them into three sub-families as EXPA, EXPB, and EXPLA. Analysis on gene structure and sequence feature shows that the gene sequence of maize is actually highly conservative. Physical positoning of Chromosome shows that the distributions of all the 88 genes on 10 Chromosomes are random except the eighth Chromosome. To analyse the gene duplication of maize, we found 5 segmental duplications and 5 tandem duplications, which mean a joint function of these two duplications in the evolution process of gene expansion of expansin gene.2. We used three ways (EST, Semi-quantitative PCR, gene chip) to analyze the expression pattern of the 88 ZmEXPs, and detected the endosperm specifi-expression of genes in maize. Finaly, endosperm specifi-expression gene ZmEXPB13 was selected as target gene, and it was cloned from B73 line in maize. It was detected that the ZmEXPB13 protein was located in cell wall by the analysis of instantaneous conversion.3. Constitutive promoter (35S) and endosperm-specific promoter (GLU) were used to promote ZmEXPB13 gene, and the over-expression vectores were constructed. Then the two kinds of vectores were transformed into rice. All the transgenetic lines were detected by PCR and GUS assay. Respectively, we got 16,18 positive transgenic plant lines. Six lines (13-3,13-10,13-13; 13G-3,13G-5,13G-10) of single copy transgenic lines were detected by the Southern detection of the To positive transgenic plant lines. The six lines of transgenic lines were used to study the function of ZmEXPBl3 in future.4. The character analysis of the 6 lines showed that the length and the weight of the matrure grains of transgenetic plants were significantly improved compared with control plants. The cell size and cell wall thickness increased significantly among diffrent trasgenetic lines by the analysis of paraffin and transmission electron microscope in sub-cellular level. And there were clear intercellular layers in trasgenetic lines. A significant increase in the column ensheathing cells and cortical cells was also detected in the young roots in transgenic plants. It indicated that ZmEXPB13 enyoyed the function to induce cell expansion and cell enlargement. When ZmEXPB13 was over-expressed in endosperm, it could promote endosperm cell size increased, and lead to the size and weight of seed increased. It might play important roles in the progresses of increassing crop yields.5. The promoter regions of all ZmEXPBs contain one or more ABA-or GA-response element by analyzing the cis-elements of the upstream 2kb promoter regions of ZmEXPBs. The results indicated that the two plant homones might play important roles in regulating the expression of ZmEXPBs in plant development progresses. The expression parttens of the ZmEXPs expressed in endosperm under ABA and GA3 treatments were studied by qRT-PCR. It showed that all these ZmEXPs were sharply upregulated after ABA or GA3 treatment. The results will provide important reference information for the study of the mechanism of protein expansin.In conclusion, we have identified 88 ZmEXPs genes altogether. The comprehensive and systematic analysis of this gene family included analysis on gene structural features, evolutionary relationships and various expression modes. And the endosperm-specific expressed gene (ZmEXPB13) was identified and cloned. It showed that ZmEXPB13 had a significant effect in the function of inducing cell size increased, improving seed size and wheight by the analysis of its biological and functional. Besides, to deal with endosperm of maize by GA or ABA, we found that the amount of expressed ZmEXPs is obviously increased, which means an important regulatory function of GA and ABA in the expression of expansin gene along the process of seed development. The results will provide valuable information for the further revelation of the functional mechanism of expansin and improve crop production by the way of genetic engineering.