Cloning And Characterization Of A CDNA Encoding Putative Neutral/Alkaline Invertase From Rice (Oryza Sativa L.)

Rice is one of staple grain crops, now it is a kind of modern plant in molecular biology. Go along with finishing of rice genomic sequencing, it becoming an arduous task to explain and make use of these data. Make full use of rice genes would help us to understand essence of life, and have important significance to raise yield, improve quality. Leaf is an important organ to nutritious growth, which is not only a place of transpiration, but also a place of photosynthesis. Leaf is source of energy and material. There are a lot of distinctive express genes in leaf. These genes control many metabolize. It is very important to cloning and using these genes.We use Qryza sativa L. as subject of experiment, and leaf as taster, root as driver, by means of suppression subtractive hybridization (SSH), acquired many fragments that have different expression in leaf and root.Isolated two fragments that have high expression in the leaf of rice show homology to invertase gene, and use the two fragment search GenBank, then obtain genomic sequence of rice invertase gene. Primers were designed to obtain its complete cDNA. Sequencing indicates that Osinv (Qryza sativa invertase) contains 1937 bp which encoding a putative protein with 627 amino acids. Translate region is 1884bp, 5' -UTR (Untranslated Region) is 9 bp, 3' -UTR is 44 bp. The gene is locates at first chromosome of rice. Full length is 3700bp, make up by six exons. Sequence alignment shows that the deduced protein have high homology to neutral/alkalin invertases with other plants, and show 80% identi ty to D. carota and A. thaliana, RT-PCR was performed to find different of leaf, root, anther and young panicle. Furthermore, check the expression difference of stress.Main solutions in the paper are: 1. Obtained fifty-three EST by means of suppression subtractive hybridizationfrom rice leaf, and two fragments belong to rice invertase gene which lengthare 323 bp and 320 bp.2. Separated and cloned complete cDNA sequence of rice invertase through electronic clone and PCR from rice leaf. Translate region of this gene is 1884bp, encoding a putative protein with 627 amino acids.3. The rice invertase gene expresses differently in different organ, high expression in leaf, but no significant different expression in root, antheri -and young panicle.4. RT-PCR was performed and revealed that the expression of Osinv is accelerated after stress treat (such as salt and cold).