Cloning And Functional Analysis Of Flower And Fruit Development Related To Genes Of Seedless Litchi

Litchi chinensis Sonn is one of the speciality products in sub-tropic district of south china. It is a desirable fruit for its nice red color and delicious taste and has very high commercial value.. Hainan seedless litchi is an excellent variety with the high seedless rate of 95%. Unfortunately, the rate of bisexual flowers is very low and their fruits are mainly developed from the female flowers, and the three kinds of flower (bisexual flower\ female flower\male flower) could not flowering at the same time, which result in female flower poor pollination and low fruitbearing percentage. Moreover the seedless rate is unstable and was affected by the temperature during flowering stage. The rate of seedless fruit is high when the temperature is below 22℃ and it becomes low when the temperature is over 22℃ . Hainan seedless litchi's extension and planting area is seriously affected by its poor stability of seedless rate and therefore resulted in its lower commercial value. Many studies indicate that a majority of MADS-box genes are involved in controlling the flower development whereas the minority is related with embryo development. Seedless fruits would be obtained when the MADS-box gene associated with embryo development was mutated. Studies in histomorphology of litchi show that seedless litchies are induced by their embryo abortion. So the study of isolating the MADS-box genes related to flower development and fruit setting genes of hainan seedless litchi has significant importance. This paper represents the study that tries to investigate the molecular mechanism in fruit setting of litchi seedless fruit.The major results of the study are as follows:1. Flower development related to MADS-box gene in Seedless Litchi was isolated by using RT-PCR and 3'-RACE (3' rapid amplification of cDNA ends) approaches and two degenerative PCR primers against conserved sequences in the MADS-box and in the C-terminal region of several plants. The gene was named as LMADS1 and itslanding number is AY705793 on NCBI-GenBank. LMADSl contains a typical MADS-box and a K-box, which shows that it belongs to MADS-box gene family.2.Northern and Southern blots were probed with DIG-labeled the unconserved 3'-terminal cDNA sequence of LMADSl and to analyze its copies in seedless litchi genome and its expression patterns. The results show that it exists in one copy in genome and is only expressed in female and male flowers, but not expressed in leafs and in developing fruit. So we conclude that LMADSl gene is not involved in controlling litchi seedless fruit formation.3. The plant expression vector pCAMBIAUOl was digested by BglII and Bst EII and the GUS gene was removed, the cDNA sequence of LMADSl gene was put to where the GUS gene is located. Then the pCAMBIAUOl vector was sequenced and approved that the LMADSl gene was linked to the right place. The pCAMBIAUOl vector containing the LMADSl gene was transformed to Arabidopsis by using Agrobacterium tumefaciens as medium. Sixty positive lines were obtained through hygromycin screened . Seventeen lines were detected by PCR approach and thirteen were found positive. Then six lines randomly chosen were detected by genomic DNA Southern blot. The result shows that four lines were found positive, which indicates LMADSl gene was successfully transformed to Arabidopsis thaliana's genome.The transgenic lines of Arabidopsis thaliana were observed and found no significant difference between transgenic plants and wild plants in flower development process, flower identity and fruit identity.4.Using the seedless fruits as Tester, the seeded fruits as Driver, a subtracted cDNA library of seedless litchi fruits was constructed by using Suppression Subtractive Hybridization approach. The library contains 381 clones in all. Sixty positive clones were obtained by using the reverse Northern dot- blot to screen the cDNA library. Nucleotide sequences of seventeen randomly chosen positive clones were sequenced and found ten are different sequences. Subsequently seven longer sequences were analyzed for their homology to the genes in GenBank through BCNI-BLAST and found that only one sequence has significant homology identified to sequences in Genbank whereas the other six have not. It is speculated that they are new genes or the 3'untranslated regions sequences of genes since the 3'untranslated regions varies markedly. Seven randomly chosen positive clones were detected by using Northern blot for further...