Isolation And Functional Analysis Of Indica Specific Protein (ISP)

Rice(Oryza sativa L.), one of the most important food crops in the world, is the staple food for about half of the world people. At present, main cultivated rice varieties in our country can be divided into two subspecies, including indica and japonica. Indica-japonica intersubspecific hybrid F1 possessed huge heterosis. Breeding new rice varieties and strong combing ability hybrid rice parents by indica-japonica hybrid is an important way to achieve high yield breeding in rice. Therefore, this study used proteomic technology to investigate differential expression of proteins between indica and japonica, and to further explore the molecular mechanism of rice indica-japonica differentiation. It will not only be helpful to clarify the origin and evolution of the rice, but also provide a theoretical reference for utilization of indica and japonica subspecies heterosis, and has an important practical significance. The main contents and results of this study were as follows:(1) This study analyzed protein expression profiles of 20 conventional rice yellow seedlings(10 indica varieties, 10 japonica varieties) by two-dimensional electrophoresis and mass spectrometry, and a protein specifically expressed in indica rice varieties was found. So this protein was named as Indica Specific Protein(ISP), which was a salt-induced protein(salT) by mass spectrometry. Sequence analysis showed that ISP gene is consisted of two exons and an intron.(2) The full-length cDNA sequence of ISP gene was obtained by RACE technique. In the ISP gene region and its upstream and downstream sequences of Nipponbare(O. sativa L. ssp. japonica) and 93-11(O. sativa L. ssp. indica), 75 single nucleotide polymorphisms(SNPs) and 15 insertions/deletions(InDels) were identified after comparison. This indicated that the gene had a faster evolution rate, and could be related to rice indica-japonica differentiation. Bioinformatics analysis indicated that ISP was an unknown function protein. Homologous sequences were also found in corn, sorghum, wheat and pineapple, and phylogenetic analysis results suggested that these homologous proteins formed two evolutionary branches in evolution.(3) Transcription phase analysis suggested that ISP gene had diggerentiation expression both spatially and temporally. We speculated that it was regulated by the light condition as its expression level in yellow seedlings was higher than green seedlings. In addition, subcellular localization of ISP protein in the tobacco showed that ISP protein was located in the chloroplast. Reasonably, the ISP protein may be involved in light regulation pathways, but the underlying mechanisms still need further investigations.(4) ISP gene overexpression and RNAi vectors were constructed, which were subsequently transformed into Nipponbare plants, and then transgenic rices were obtained. The leaves of ISP gene overexpression transgenic plants in the mature period could be able to keep green through phenotypic observation, while the leaves of wildtype and RNAi transgenic plants turned yellow and wilt. This indicated that ISP gene could delay the senescence of rice leaves, but how ISP gene regulated the leaf senescence still remains further investigations.