Cloning And Expression Analysis Of Candidate Genes Related To Cytoplasmic Male Sterility In Pepper

Cytoplasmic male sterility (CMS) has been an ideal system to produce hybrid seed of pepper (Capsicum annuum L.).In order to elucidate the molecular basis of CMS in pepper, we use homology based candidate gene method and some other molecular biology technologies to research three sets of cytoplasmic male sterile pepper obtained from distant hybrid. The main results were as follows:1. Establish an extraction method of mitochondrial DNA(mtDNA)from pepper young leaves. We adopted differential centrifugation and high salt solvent extraction method to collect the mitochondrial from young leaves of pepper. Then the mitochondrial DNA was extracted by using SDS and Proteinase K. The protocol do not need etiolated seedlings and have low requirements in apparatus and reagent compared with previous methods. The extracted mtDNA have a good quality to be used in further research by gel electrophoresis and spectrophotometer detection.2. A specific mitochondrial DNA fragments termed cms330 was amplified from three sets of male sterile pepper material based on homology based candidate gene method. The sequence analysis revealed that cms330 has a 99% homology in nucleic acid sequence with the orf456 which had been proved to play an important role in male sterile phenomenon of pepper. It reflected that they might have the similar male sterile mechanism.3. A differential 3 kb DNA fragment was obtained from three sets of male sterile pepper by inverse PCR and long primer. The further sequence analysis indicated that the part of the upstream and downstream sequences of cms330 exhibited a higher than 85% homology with NADH dehydrogenase gene. The gene recombination might take place between cms330 and NADH dehydrogenase.4.The expression of cms330 was investigated by RT-PCR in three sets of male sterile pepper. The result showed that cms330 RNA fragment can be detected in the three sets male sterile materials and expressed in the flower and leaves in pepper.