| Maize is the most widely planted crops in the world, as well as the highest-yielding cereal crops over the past decade, which is higher than rice and wheat. Maize plays an increasingly important role in human life. It is not only the main food crops, but also the main source of starch in production. Starch can be divided into amylose and amylopectin based on different structures. These two kinds of starch play a vital role in various fields. Generally, the content of amylose in maize is about 20% while the content of amylopectin is about 80%. In order to improve the balance of the contents of starch, research on biological metabolic pathways of starch is highly needed. This study may extend previous observations made in starch metabolism, and effectively control synthesis of starch as well as improve the starch quality. In this study, starch synthesis genes were screened by genome-wide association analysis, and then expression pattern analysis was conducted on these genes. In addition, the function of ZmbZIP91 in the synthesis of starch and mechanisms involved were investigated by gene overexpression. The results are as following:(1) The association analysis was performed between the extreme traits of starch and the SNP data of 87 resequencing materials. The same analysis was carried out among traits of amylose and total starch with 36 transcription factor genes screened by transcriptome sequencing. Seven genes were obtained, and 6 of them were performed expression pattern analysis. A total starch and amylose synthesis related gene was screened. This gene only expressed in endosperm, and the amount of expression increased with the days after pollination.(2) We performed histochemical and quantitative β-glucuronidase(GUS) analysis of the Zea mays legumin1(ZM-LEGF) gene promoter and detailed detection of stably transformed rice expressing the GUS gene under the control the promoter of ZM-LEGF(pZM-LEGF) and its truncated promoters throughout development. When the promoter sequence was truncated, the location and intensity of GUS expression changed. The results suggest that the sequence from-140 to +41 is a critical region that confers the expression of the entire promoter. Truncation of pZM-LEG(3’-deleted region of pZM-LEGF) markedly increased the GUS activity, with the core cis-elements located in the-273 to-140 regions, namely, pZM-LEG6. Detailed analysis of pZM-LEG6::GUS T2 transformant rice seeds indicated that this gene was not expressed in seeds until germination, indicating that pZM-LEG6 is a vegetative-tissue promoter.(3) Subcellular localization experiment showed that ZmbZIP91 and fusion protein GFP located in P-body. No nuclear localization sequence and signal peptide was found by online analysis, indicating that this gene cannot express in nuclear and unable to regulate the expression of functional genes. The results of yeast single hybrid showed that ZmbZIP91 interact with known starch synthesis genes of maize, including ZmAGPL1, ZmBEIIa, pZmISA2, ZmSSIV, ZmGBSSIIb and ZmSSI. These results suggested the gene might be involved in starch synthesis.(4) No significant difference was detected in transgenic rice of ZmbZIP91 overexpression except the change of seed. By analysis of 17 starch synthesis genes, we found that the expression of seeds in transgenic rice was lower than wild type. Study on external phenotype of seed showed that the length of seeds in transgenic rice was almost unchanged, but the width was significantly less than wild type and empty vector, reduced by 12.28% compared to wide type. Thousand seed weight was also less than wild type and empty vector, reduced by 16.16% compared to wide type. For the analysis of starch content, the contents of total starch and amylase of seeds in transgenic rice were significantly lower than wild type and empty vector, reduced by 6.96% and 21.83% respectively compared to wide type. Analysis of the starch structure by paraffin method showed that no significant change of seeds was found between transgenic rice and wild type under invisible light. However, under ultraviolet light, yellow light was observed in the seeds of transgenic rice while none can be detected in wild type. Scanning electron microscopy detecting showed that the starch granules at the middle, the abdomen and the back in the seeds of transgenic rice were significantly smaller than wild type and empty vector. It suggested that ZmbZIP91 may play a negative control in the process of starch synthesis in rice.(5) The analyzing results of the mutantion(m-91) showed that the ear length, anther, ovary, glumes, pilose and seeds were significantly greater than wild type. The increase of glume tips was the most obvious, which increased by 164.11% on average compared to wild type. Comparative analysis of the length and the width indicated that the length of mutantion was significantly higher than wild type and the width of mutantion was slightly higher than wild type, increased by 22.01% and 8.12% respectively. Thousand seed weight was also higher than wild type, increased by 22.99% on average. Scanning electron microscopy detecting of the shelled seed and the starch structure showed that the skin was wrinkled, separated from the endosperm. Chalkiness was observed in the central part. Starch grains were polyhedral. Most of the starch grains and proteosomes were loosely arranged, and a small amount of starch grains formed complex embedded in the proteosomes. Analysis of total starch and amylose showed that the content of amylose reduced by 24.25% compared to wild type. Meanwhile, the change of total starch was not obvious, only reduced by 2.95%. These results suggested ZmbZIP91 may affect the enzyme activity and gene expression related to these traits. |
