Construction Of Suppression Subtracted CDNA Libraries Of Betula Platyphylla In Flower Phase And Cloning Of Genes Related To Flower Development

Betula platyphylla, a kind of pioneer tree species in Northeast and Inner Mongolia of China, is widely used in construction, furniture, and paper production. In order to shorten breeding period and accelerate breeding process, a breakthrough has been made in early flower inducing in recent years. However, little is known about the background of gene expression in flower phase of B. platyphylla, but several genes related to floral development were successfully cloned in Betula pendula in latest years. To supplement more useful information in this field, the following work has been done based on the previous study on reproductive biology of B. platyphylla.After B. platyphylla inflorescence were sampled in each developmental stage, suppression subtracted cDNA libraries based on male and female inflorescence were constructed using PCR-Select? cDNA Subtraction Kit by SMART (Switching Mechanism Along 5' end of RNA Transcript) strategy. By multiple blast analyses and reckon, the redundancy is 12.14% in the forward library of female inflorescence, 15.48% in reverse library of female inflorescence, 29.30% in the forward library of male inflorescence, 27.85% in reverse library of male inflorescence. After repeated ESTs are excluded, expect value is set to e^-10, rDNA contamination and mosaic cloning is eliminated by blastX analyses, there are 65 ESTs in the forward library of female inflorescence, 71 in the reverse library of female inflorescence, 68 in the forward library of male inflorescence, 65 in the reverse library of male inflorescence. By further treatment, 126 ESTs are acquired in the suppression subtracted cDNA libraries of female inflorescence, 115 ESTs in that of male inflorescence. There are some different ESTs representing the same kind of blast result in the libraries, which were showed corresponding to different sequences of the same gene or represent different members of the same gene family. Seldom redundancy occurred between male and female inflorescence suppression subtracted cDNA libraries each other by comparison. According to GO classification system, the ESTs acquired in the libraries are involved in metabolism, cell growth and division, cell architecture composition, transmembrane transport, signal transduction, stress response, transcriptional control,, translation, protein degradation and other vital process. There is rDNA contamination to some extent in the sequencing results since the total RNA are used as the original material for constructing libraries. The Blast results showed that some ESTs obviously related to flower development are contained in the libraries, which will be the material resouce for further study on floral development of birch.Referred to the published sequences of B. pendula in the public database, 4 genes (BpMADS1, 3, 5, and BpSPLI) related to floral development are cloned from B. platyphylla byRT-PCR. Sequences analysis showed that variation occurred both in nucleic acid and protein sequences between these two birch species. The ESTs of three genes don't exist in the libraries, the cause of this phenomenon is inferred in this dissertation. With two ESTs in the suppression subtracted cDNA libraries used as the original materials, two new genes (BPSPL2 and BPAGL2) related to flower development were cloned by RACE, then their phylogenetic positions were inferred by the tool of Tree TOP.A real-time quantitative PCR system is established using the ESTs in the suppression subtracted cDNA libraries of 4 genes related to flower development, which showed they all temporally expressed during inflorescence development, and their probably functions are primarily deduced according to their expression modes.