The Effect Of Circadian Clock Gene LHY?CCA1 On Vegetative Phase Change In Arabidopsis Thaliana

The growth and development of higher plants is a complex process.Developmental phase of higher plant is composed of two stages:vegetative growth and reproductive growth.The vegetative phase can be divided into a juvenile stage and an adult phase.The transition from juvenile to adult phase is termed as vegetative phase change(VPC).VPC is an indispensable process in the growth and development of plants,which is regulated by genes expression.VPC is associated with change in plant morphology,cytology and the molecular level.The onset of the abaxial trichomes is often regarded as a marker of VPC,while the degree of bulging of shoot apical meristems can also be used as a dividing line from the juvenile stage to the adult stages.Moreover,VPC is regulated by the expression level of microRNA-miR156 and its target gene SPL3.There is a circadian clock control mechanism in the plant.In the long-term evolutionary process,the impact of the circadian clock with the environment and day and night alternatively,completing the growth and development process of the plant.The inner circulation mechanism regulating the biological clock is called the circadian clock.Although the study of circadian clocks is relatively thorough,the role of circadian clock genes in VPC,which is less understood.In this study,Arabidopsis thaliana circadian clock gene double mutant lhyccal and LHY?CCA1 overexpressing plants were used as materials.Morphological,cytological and molecular level investigations were carried out to analyze the effects of circadian clock genes LHY and CCA1 on VPC.In this experiment,the total RNA of wild type Arabidopsis thaliana was firstly extracted,and the full-length coding sequence of LHY and CCA1 was amplified by RT-PCR,and its length of coding sequence was about 2 kb.The plant expression vector pCAMBIA-3301-35S::LHY and pRI101-AN-35S::CCA1 were successfully constructed.After transformed to wild-type via dip flower,the screening of resistance was performed by Basta and kan respectively,and finally transgenic positive homozygous plants lines were obtained.Comparing the double mutant and the overexpressing plant with the wild type at three levels were performed respectively.The results showed that the double mutant lhyccal had a shorter growth cycle than wild type,while the LHY and CCA1 overexpressing plants had longer growth cycle than wild type.The wild type plant has bolted on the 20th day,while the double mutant lhyccal has bolted on the 15th day.The LHY and CCA1 overexpressing plants has bolted on 33 d and 41 d separately.In wild type,double mutant lhyccal,LHY and CCA1 overexpressing plants,onset of abaxial trichomes,as well as the increasing of leaf basal angle and leaf length-width ratio,was on the sixth rosette leaf(the 15th d),the fifth rosette leaf(the 10th d),the seventh rosette leaf(the 19th d),and the eighth rosette leaves(the 21th d),respectively.Meanwhile,shoot apical meristem was obviously bulged,and the expression levels of miRl56 and SPL3 decreased or increased dramatically.These results indicate that vegetative phase change of double mutant lhyccal was earlier than that of wild type,while that vegetative phase change of LHY and CCA1 overexpressing plants was delayed.