Large-Scale EST Sequencing Of CCRI36 CDNA Library And Pollen Development Specific Gene Clone

Large-scale EST Sequencing of cDNA library has been proved to be a rapid and efficient method for identify the novel genes,obtain EST and functional genomes research.In order to elucidate cotton inflorescence development and exploit early maturity gene resources,we carry out high-throughout 3'EST sequencing the normalized cDNA library of flower development of CCRI36 . A lot of useful ESTs were captured and pollen specific gene was discovered through bioinformatical analyse. It is of significance to study pollen specific gene for elucidate the mechanism of Pollen Development and the application of male sterile lines. Through EST sequencing and pollen specific gene clone,the main results were as follows:1.Through large-scale EST sequencing of cDNA library obtained 7072 high quality ESTs, 6246 Unigenes.After removing vector sequence the average length of clear EST is 483.5bp.The repeat rate is 11.36% of this sequencing . This made up for the lack of cotton flowering-related EST At present. This lay a good foundation for cotton functional genomics research and Important flowering Genetic resources exploitation.2.Bioinformatics analysis of the EST data some important genes that control flower development and five pollen-specific genes were discovered .3.Five cotton chromosome walking library were Constructed,this will play an important role in promoter, introns and other flanking sequences cloning.4.A pollen-specific gene has been cloned named GhAS1. GhAS1 promoter and the downstream regulatory sequence 3002bp in all has been cloned, Genbank registration number (HM021767).5.The expression patterns of GhAS1 gene has been clarified By QRT-PCR Analysis. The results showed that this gene is pollen-specific,This lay a good foundation for utilization of gene and pollen specific promoter.6.Molecular function prediction shows GhAS1 encoded protein has a typical signal peptide, transmembrane, hydrophilic ; Subcellular prediction localizated the gene to extracellular, functional domain analysis showed that this gene belongs to the family of pollen allergens;Homology alignment analysis suggested that gene function is to promote pollen germination,high-profile expression late in the of pollen development.7.Based on pollen specific gene GhAS1 isolated from Gossypium hirsutum,we constrcted a universal RNAi vector pCALOXRI. In this vector expression cassette regulated and controlled by Cre/loxP systerm,driven by pollen-specific promoter LAT52.LAT52promoter,GhAS1 sense,AF intron, and GhAS1 antisense had been construed an RNAi expression cassette.The cassette flank- ed by two lox sites in a directed orientation so that can be deleted by Cre recombination enzyme. This systerm set Cre/loxP system efficiently recombination, tissue-specific promoter control of gene expression accurately, RNAi a high degree of specificity and high efficiency three advantages in one. The purpose is to build a universal system for examinating research pollen development tal genes, and lay a good foundation for create artificial male sterile lines and restorer lines .8.Through the pollen tube pathway transgene, For GhAS1 gene overexpression and RNA interference research,two receptors materials zhong99668 and CCRI36 were harvested 1625g and 497g seeds respectively. Field identification by kanamycin in progress.