Cloning And Expression Characterization Of GhMYBL In Cotton

Cotton is a mainstay of economy in China, which is the largest cotton producer and consumer around the world. Due to the negative linkage between yield and production of cotton, it is very difficult to synchronously improve both of them by traditional methods. Gene engineering provides an effective strategy to improve the yield and quality of cotton. For this purpose, it is of significance to clone gene related grown regulation and to elucidate the molecular basis of grown development.MYB genes constitute the largest gene family oftranscription factors in plant. The common feature of MYB transcription factors (TFs) is the highly conserved DNA-binding domain (MYB domain). Most plant MYB genes belong to the R2R3-MYB subfamily and are involved in a variety of biological functions. They have been shown to play important roles in the regulation of phenlpropanoid metabolism, the control of development and determination of cell fate and identity, as well as plant responses to hormones and environmental factors. So, the MYB gene family is of great importance in transcriptional control in higher plants. Now most researches about MYB TFs in cotton were focus on development of cotton fiber. Little was reported about the MYB TFs in anther. So it is significient to do further research in this field.We isolated 30 TFs genes from cotton flower cDNA library. One gene which belongs to MYB TFs family and is expressed specifically in cotton anther was chose to make further reseatch by bioinformation and RT-PCR motheds. And it was defined GhMYBL. The main results are as follow.1. Cloning and characterization of one GhMYBL gene.One gene defined GhMYBL was choes by bioinformation and RT-PCR methods. Sequence analysis shows that GhMYBL encoding a protein with 210 amino acids contains two MYB domains and is thought to be a member of the R2R3 MYB gene family. Expression pattern by RT-PCR indicates this gene expressed specifically in anthers, suggesting that GhMYBL probably involves in the anther development.2. GhMYBL expressed specifically in cotton anther.Cotton total RNA was isolated from different tissues of cotton, different developmental period of cotton fiber and ovule by hot-phenolic method. We primarily make use of real-time RT-PCR methods to analysis the expression pattern of GhMYBL gene. Then northern hybridization analysis was used to confirm the expression pattern ofGhMYBL. Both results showed that the GhMYBL gene was expressed specifically inanther.3. Vector construction and cotton transformationThe GhMYBL gene was cloned into the pBI121 vector to obtain over expression vector and suppression vector respectively. The results of PCR reaction and cutting with enzymes showed that the target gene was already successfully fused with vector. The reform vectors were transformated into cotton by using of the DNA transformation system medimated by Agrobacterium (LBA4404). The transfer resistant calli were subculture 12 monthes. Now parts of the calli are induced into somatic embryos.