Cloning And Characterization Of SRK-Binding Protein Gene And Study On Its Molecular Mechanism In Self-incompatibility Signaling Process

Brassica crops occupy the largest cultivated areas and produce the highest yields among the vegetables and oil crops in our countryside. It is one of the most prevalent crops in the utilization of heterosis and have been regarded as model plants in the process of researches on their molecular mechanism of self-incompatibility and breeding and application of male sterility.ARC1, THL1 and THL2, the substrate protein genes of S receptor kinase, were cloned through a series of methods of molecular biology such as PCR, RT-PCR, DNA cloning and sequencing. The resultings sequences were highly analysed by using the related biosoftwares on Internet, providing new insights in the field of the molecular mechanism of self-incompatibility in plants. The major results are as followings:1 . The method of CTAB was specifically improved for the extraction of genomic DNA and total RNAs from Brassica oleracea L. and Brassica napus L.2. ARClBoler gene was amplified by PCR with template genomic DNA and cDNA from roots, leaves, petals, stigmas and ovaries during bud stage in Brassica oleracea L.(200110197) and Brassica napus L.(Y578-2). The DNA sequencing shows that ARClBoler gene shows 94% identity to the sequence of ARC1 gene of Brassica napus L.. There is only an 1983 bp ORF in ARClBoler gene that encodes 661 amino acids. The amino acid sequence shows 85% and 52% identity to those of ARClBnap gene and ARC1At gene respectively. These three proteins contain two common clif domains, Ubox and Arm Repeats. Ubox is putative domain of dimeration of ARC1 protein or the binding domain of next element. In the C-terminal of ARClBoler protein and ARClBnap protein, there are five arm repeats, in contrast to only one in ARClAt protein. This difference potentially determines the discrepancy of function of ARClBoler, ARClBnap and ARClAt. The enzyme digest analysis shows that the arm repeats of C-terminal are conceivably conservative domain. In ARC1 protein, there are some active sites including N-glycosylation sites, cAMP- and cGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites, Tyrosine kinase phosphorylation sites, N-myristoylation sites, amidation sites and Leucine zipper pattern. It probably take part in the signaling process of self-incompatibility. From the three dimensional structure of ARC1 can be found a wide groove and a narrow groove, which may be the binding sites of other proteins.3. THL1 gene was also amplified by PCR with template genomic DNA and cDNA from roots, leaves, petals, stigmas and ovaries during bud stage in Brassica oleracea L.(200110197) and Brassica napus L.(Y578-2). The DNA sequencing shows that THL1 gene occupies a fragment of 719 bp in gDNA, which occurs in cDNA with a length of only 430 bp. The alignment analysis between gDNA sequence and cDNA sequence of THL1 gene shows that THL1 gene contains two introns of 207 bp and 82 bp respectively. Its cDNA encodes 117 amino acids. In THL1 gene, there are some famililar enzyme digest sites including BamHI, Hind , Sac I and Sal I .The sequence of THL1l from Brassica oleracea L. shows 99% identity to the sequence of THLl from Brassica napus L. There are some phosphorylation sites and an active site of thoiredoxin h members consisting of CPPC in THLl protein. Although these phosphorylation sites are silent in the signaling of self-incompatibility, it possibly play roles in other biological processes.4. THL2 gene was finally amplified by PCR with template genomic DNA and cDNA from roots, leaves, petals, stigmas and ovaries during bud stage in Brassica oleracea l.(200110197) and Brassica napus L.(Y578-2).The DNA sequencing result shows that the genomic gene of THL2 is approximately 900 bp from Brassica oleracea L. in contrast to that of 850 bp from Brassica napus L.. The cDNA of THL2 from Brassica napus L. is 447 bp. THL2 gene contains two introns with wide divergences between Brassica oleracea L. and Brassica napus L.. However, the deduced amino acid sequence of THL2 protein f...