Functional Analysis Of ESP2, A Gene Controlling Panicle Enclosure In Rice

Currently, most of Indica CMS in use have different degrees of sheathed panicles. We acquired a fully panicle sheathed mutant from Indica Ming Hui 86, named esp2. Our previous studies showed that, ESP2 affects the development of the uppermost internode; results of gene mapping and complementary experiment indicated that ESP2 is a phosphatidylserine synthase (PSS) gene. On this basis, we further analysed the other agronomic traits of esp2, characteristics of ESP2 gene structure, expression and function of the gene in the mutant, and subcellular localization of the protein.The main results are as follows:(1) In addition to affecting the development of uppermost internode and spike in rice, the ESP2 gene also influence the development of the reciprocal three leaves in different degrees. But it has no significant effect on rice growth period, leaf color, tiller number or other important agronomic traits.(2) Bioinformatics software analysis indicates that ESP2 contains a PSS functional domain, which catalyzes phospholipids and nucleophilic donor to synthetize PS by transesterification reaction. The conserved sequence of the enzyme protein, which is fully consistent with PSS1 of hamster, begins from the initiation codon and contains 280 amino acids, including 1 phosphate binding sites,8 catalysis active sites and 6 Stability regulatory sites. So we proposed that ESP2 is a kind of phosphatidylserine synthase (PSS). There are mainly two kinds of PSS reported in plants:BE-PSS and CD-PSS, but it needs further research and analysis to know which kind of PSS that ESP2 belongs to.(3) Bioinformatics analysis showed that the protein ESP2 is mainly located in the cell membrane. An Subcellular localization vector of pCX-DG-ESP2 was constructed and transferred into the onion epidermal cells by Agrobacterium infection method. The results showed that ESP2-GFP fusion protein expresses specially in the cell membrane, indicating that ESP2 is likely to be a membrane protein.(4) Sequence analysis of the esp2 transcription product showed that 5287 bp was inserted at the end of the fourth exon, causing the third and fourth intron retained in the ORF, resulting in a frameshift mutation and a terminator TGA occurred at the 97th codon. So the function of the gene esp2 was completely lost, and esp2 should be a null-allelic mutant.(5) sequence analysis of ESP 2 in several rice varieties (Indica and Japonica) showed that DNA sequence of the gene is the same, but the length of ORF is different. The 495bp after the initiation codon of ORF is exactly the same in these species, but because of the different splice sites of the sixth intron, the length of its ORF is different. According to bioinformatics analysis results of ESP2 and normal phenotype of these tests rice varieties, we proposed that the key domain (165 amino acids) determining the function of ESP2 protein is highly conservative in the rice evolution, despite the ESP2 ORFs of different varieties are changed.