Clone And Expression Analysis Of Maize Endosperm Specific Promoter

There are a lot of genes relevant to starch synthase, prolamin synthetase, glutelin synthase in maize endosperm. Parts of these genes are regulated by the endosperm specific promoters, and only expressing in the endosperm. In this study, we have cloned the promoters of starch synthase gene ZMGBSS and glutelin synthase gene ZMGT1, SC3 and prolamin synthetase gene ZEJN, BN15D14A from the inbred line 18-599 Red DNA. They are named to GBSS, GT, SC, ZEIN, BN. At the same time, the expression of its tissue-specificity and induced-factor were analyzed by RT-PCR and qRT_PCR.The activities were also detected by transient expression. The results are as follows:1. About 2000bp length fragments of endosperm-specific promoter GBSS, GT, SC, ZEIN, BN) were successfully amplifyicatied by high fidelity polymerases system KOD Fx, and the cloned fragment were recycled and sequenced. The sequence similarity was 97%,99%,99%,99% and 99% in comparison to B73 genome in the NCBI blast respectively.2. RNA were extracted from different organizations in different periods from maize inbred lines 18-599 red. Semi-quantitative PCR were used to analyse the expression specifitility of ZMGBSS、ZMGT1、SC3、ZEIN、BN15D14A.The expression of ZMGBSS and ZEIN are particularly high in endosperm.3. The qRT-PCR analysis were used to investigate the expression of ZMGBSS、ZMGT1、 SC3、ZEIN、BN15D14A in endosperm after the hormone-induced.The endogenous expression activity of ZMGBSS and ZEIN in endosperm have significantly improved by ABA treatment.At the same time, the endogenous expression activity of ZMGTI and BN15D14A in endosperm also have been improved by sucrose treatment,.4. Transient expression vectors have been successfully builded with promoter GBSS, GT, SC, ZEIN and BN. They are pGBSS-Luc, pGT-Luc, pSC-Luc, pZEIN-Luc and pMBN-Luc. The internal pUbi-Gus and controlled pUbi-Luc transient expression vectors were also constructed in this study. And transformed to endosperms, embryo of 14day after pollinating, leaf and root of 10day after germinated. And their expression were detected according to Luc and GUS.The activities of promoter GBSS、ZEIN and BN were relativity high compare to SC and GT,but the activity of cloned promoters were all much more lower than Ubiquitin.5. Endosperms traeted with glucose, sucrose, ABA, GA for 4 hours in vitro were used as transform acceptor. And their expression levels were detected by Microprojectile bombardment with the transient expression vectors. The results confirmed that the activities of GBSS, ZEIN were obviously enhanced by ABA-treatment, and the activities of GT, BN were also enhanced by sucrose-treatment, but the activities of SC were restrained by all thetraetments in this study.