Construction Of Subtracted CDNA Libraries Of Ovules From Pinus Tabulaeformis Carr In The Period Of Free Nuclei

Explore the female gametophyte development in the expression of gene regulation mechanism development is important for revealing the molecular mechanism of plant. The female sterile clone 28 is aborted because of the failure development in the female gametophyte.To explore the reasons from the perspective of molecular biology and determine the functions of differentially expressed proteins, we should screen differentially expressed mRNA associated with plant development.In this paper,using Plant RNA assistant kit can obtain high-quality total RNA of ovules by comprising four methods of total RNA extraction.The first-strand of cDNA was synthesized by using Super SMART cDNA synthesis technology,and then the second-strand was optimized and amplified.At last,the forward and reverse Subtracted cDNA libraries of ovules from the female sterile and fertile Pinus tabulaeformis Carr were constructed by suppressed subtractive hybridization.100 positive clones were randomly selected in forward library, and 75 positive clones were randomly selected in reverse library.86 effective clones in the forward library and 71 effective clones in the reverse library were got by detecting. According to sequencing these clones,72 effective sequences in the forward library and 55 effective sequences in the reverse library were obtained. Using the homology analysis of these sequences by BLASTx, the results screened some proteins which may be related to development such as cellulose synthase, glutathione peroxidase, aquaporins, Zinc finger protein, catalase, late embryogenesis abundant protein.