Isolation And Cloning Of Dominant Glandless Genes Of Cotton Using Suppression Subtractive Hybridization

Cotton is one of the most important economic crops in the world and it is the most important source of natural fiber. Cottonseed is rich in protein, fat, carbohydrate, cellulose and mineral elements and so on, yet it is underutilized due to the presence of gossypol, a binaphthyl terpene in pigmented glands throughout nearly whole plant and is toxic to human and non-ruminant animals. Breeders all over the world have been developing glandless cotton breeding. Most glandless varieties, which are easy to produce hybridization naturally in the field, and cause unsafe to eat, are due to recessive glandless genes. Dominant glandless gene of cotton is an important genetic resource in glandless breeding, thus study of these genes is important for both theory and breeding.In order to screen and clone the dominant glandless genes, a cDNA library was constructed using suppression subtractive hybridization (SSH), which could offer the material base to engineer the glanded plants that produced glandless seed. The main results were as follows:1. This study constructed dominant glandless cDNA expression library Via SSH. A dominant glandless cDNA differentiation expressed library was constructed with the cDNA of Glandless Cotton Research Institute 12 (Glandless CRI12) of the Near-isogenic line (NILs) as the Tester and the cDNA CRI12 of NILs as the Driver. The inserted fragments of 0.2~1.0 kb in the 20 randomly selected positive clones were identified with PCR analysis.2. 363 cDNA fragments were identified with reverse Northern Blotting. After sequencing, 299 qualified cDNA fragments were obtained. After cluster analysis, 147 Unigenes were abtained, including 56 contigs and 91 singlets. The analysis revealed that the library included Gossypium hirsutum chalcone synthase gene, Gossypium hirsutum gland development related protein 87-like mRNA, apoptosis related gene, oxygenase gene, ABC transporter gene and so on. Besides, 43 unknown ESTs were looked forword further research.3. The sequences of 4-FA11 and 4-FA12 were extended using rapid amplification of cDNA ends (RACE) approach. The full length of 4-FA11 and 4-FA12 were 1566 bp and 998bp, and they encoded 398 amino acids and 167 amino acid, respectively. The sequence alignment results showed that 4-FA11 had high similarity to chalcone synthase gene (CHS) in Gossypium hirsutumat and 4-FA12 had high similarity to Gossypium hirsutum gland development related protein 87-like mRNA at the level of nucleotide acids. The amino acid sequences of 4-FA11 were consistent with the CHS in Gossypium hirsutum exactly and the amino acid sequences of 4-FA12 were similar to some function unknown sequences of Populus trichocarpa, Solanum lycopersicumat, Vitis vinifera at the level of amino acids. Domain analysis revealed that the sequence of 4-FA12 contained the conserved region of copper-binding protein.