Primary Study On Cloning And Expression Analysis Of MADS-box Genes From Strawberry (Fragaria Ananassa Duch.) Fruit

The storage quality is one of the most important factors showing features of fruit. The traditionally physiological and biochemical methods to keep fruit fresh had little effect. With the rapid development of molecular biological technology, it will be a necessary way to regulate the maturation and aging of fruit in gene level in future studies. The way from ethylene synthetic only works to the variant fruit. Therefore, only finding more upstream transcription factor can solve this problem fundamentally.The cultivated area of berries of strawberry is only less than that of grape in the world. In addition, it is always less than needed. Strawberry is different from perennial woody fruit trees. It is herbaceous and ratoon fruit tree and.is also the plant reaped within the shortest period. It can be planted all of the year, and can be used as the most proper experimental material. Thereby, research on regulation in strawberry fruit ripending by gene is very meaningful—for its being a typical non-respiratory jump variant fruit trees.This paper studies on MADS-box gene of strawberry in the research mode of MADS-box, which is a multigene family.33MADS-box gene cDNA clips had been obtained from strawberry green fruit, and genes of FaMADS1 and FaMADS2 had been cloned from strawberry. We have made primer studies for expression characteristics of these two genes in all periods, in order to use these genes for fruit ripening regulation of strawberry or other non-respiratory jump variant fruit trees, or providing theoretical basis about storage quality. Specific results of the research are as follows:1. MADS-box genes had been cloned in variety of plants as an important transcription factors for regulating flower development and fruit maturation. In order to get new MADS-box genes associated with fruit maturation from strawberry, we designed degenerate primers according to the conservative region sequences of MADS-box family genes from various plants, obtaining 33 MADS-box gene fragments by RT-PCR from RNA of green fruiting strawberry. Sequence analysis showed that the fragment length were between in 137-146bp, including starting anticodon.The sequence of amino acids had a higher identity of over 87% with registered strawberry MADS-box gene. All these genetic fragments were grouped into the Arabidopsis MADS-box gene subfamily by phylogeny construction of sequences. These results indicated that there were many MADS-box genes in strawberry. Some of these genetic fragments may play an important role in adjusting fruit development, ripening even to softing.2. In order to studying MADS genes related to ripening of Fragaria ananassa, we designed specific primers form the sequence of Fv-MADS-9 gene in Fragaria virginiana and cloned the conservative part by the PCR technology, and obtained the whole gene by RACE-PCR. The GenBank registration number is HQ602763,which is named as FaMADS1.The cDNA sequence length is 1167 bp. its coding 249 amino acids, 100bp in the 5'UTR,320bp in the 3'UTR, and 30bp in poly(A) tail. The molecular mass of amino acids derived 28.7 KDa, pI for 8.51.The sequence homology comparison showed a high homology of the nucleotide sequences with MADS genes of other plant from the GenBank. It's 98% for Fv-MADS-9. Sequence analysis shows that, there are also some highly conservative area of MADS-box protein in FaMADS1 gene, such as MADS structure domain and K domain.3. Another MADS-box gene named FaMADS2 was cloned from strawberry (Fragaria ananassa Duch.) through 3'-RACE-PCR.The cDNA sequence length is 870 bp, The GenBank registration number is HQ602764, which is named as FaMADS2. It coded 180 amino acids,254bp in the 3'UTR, and 27bp in poly(A) tail. The molecular mass of amino acids derived 20.7 KDa, pI for 5.83.The sequence homology comparison showed that the exhibits a high homology of the nucleotide sequences with FcMADS-2 gene from the GenBank. It's 97% and 96% for FcMADS-1 and FcMADS-2.Sequence analysis shows that, there are also some highly conservative areas of MADS-box protein in FaMADS1 gene, such as K domain. 4. Through ative RT-PCR technology, we had analyzed the expression of FaMADS1,FaMADS2 genes in the eight different periods of strawberry fruit, the results showed that the expression of FaMADS1 was found in flower and fruit of strawberry except in flower organs. The expression levels in different stage from flower and fruit ranking low to high, with the relative content of 0,0.568,0.751,0.763,0.874,0.917, 1.099,0.837 respectively. The expression of FaMADS2 was found in all stage of flower and fruit of strawberry. The expression levels in different stage from flower and fruit ranking low to high, then to low with the relative content of 2.90,1.93,2.20,1.55,1.62, 1.99,1.44,1.43 respectively.