The Cloning And Expression Analysis Of Circadian Clock Gene ZmELF4 In Maize

The maize germplasm resources from the tropical and subtropical areas have abundant genetic diversity,and show excellent properties.Light and temperature are the major factors limiting maize cultivation and geographical distribution.Plants have developed many mechanisms to adapt to such environment changes,and the circadian clock regulation is the most important mechanisms.In order to achieve normal growth and reproduction,plant use circadian clock to sense the changes of environment factors,such as light,temperature,then adapt to different environments by generating different output rhythm.Therefore,the study of genes function of maize circadian clock component,the clock cycle system,as well as passivation sensitivity of tropical and subtropical circadian clock to photoperiod is vital to improve and make full use of tropical and subtropical maize seeds resources.Our lab has analysed photoperiod-sensitive gene of maize,and identified a number of candidate genes for maize photoperiod.In this study,a putative candidate gene ZmELF4 was cloned from the photoperiod sensitive material "18-599 white" and photoperiod insensitive material "zheng 5 8".qRT-PCR was performed to analysis the expression pattern of ZmELF4 at different growth stage of "18-599 white"under long-day and short-day conditions and to analysis the expression patterns of ZmELF4 with ZmCCA1 and ZmTOC1 in the two material under continuous light conditions.In addition,the expression levels of ZmELF4,ZmCCA1,ZmTOC1,ZmGI,ZmCONZI,ZmZCN8 were also investigated in the two materials during the period of maize floral transition under long-day and short-day condition.Finally,the over-expression vectors and transient expression vectors of ZmELF4 gene from "18-599 white" and "Zheng 58" were constructed to analysis the functions of these genes by transgene and subcellular localization by Agrobacterium-mediated Arabidopsis thaliana and Tobacco transformation,respectively.The main results are as follows:1.ZmELF4 genes were cloned from the photoperiod-sensitive material "18-599 white"and photoperiod-insensitive material "Zheng 58".Sequences alignment analysis found that the coding region of ZmELF4 gene from "Zheng 58" was intact with B73 reference sequence.However,the coding region of ZmELF4 gene from "18-599 white" lose a 37 bp sequence and cause frameshift mutant,but the predicte conserved amino acid domain was not change.The phylogenetic analysis showed that ZmELF4 gene and PsELF4 belong to ELF4 subfamily,whereas,OsELF4-like gene belongs to another branch of the EFL family.Therefore,we hypothesized that ZmELF4 may be a homologous gene of PsELF4 and AtELF4 and may has similar function with them.2.The Real-time quantitative PCR was performed to analysis ZmELF4 expression patterns in the whole growth cycle of 18-599 white under long-day and short-day conditions,and the result showed that the peak value relatively delay,the number of leaves is less,and flowing is ahead of time under short-day condition compare with long-day condition.The expression patterns of ZmCCA1,ZmTOC1,ZmELF4 at the time of transformation of maize flowering in two materials under long-day and short-day conditions were also investigated,the results showed that the expression levels of ZmCCA1,ZmTOC1,ZmELF4 under short-day condition is less than long-day condition,but there exist a little different for the strongest rhythm under the two conditions.The expression of ZmGI,ZmCONZI,ZmZCN8 is higher under short-day condition than long-day condition,so we believe that there may has a certain relationship between early flowering under short-day condition and the circadian clock output that cause ZmCONZI and ZmZCN8 up-regulation.Then,we changed the two materials growth condition into continuous light,and the expression analysis found that the rhythm cycle of ZmELF4 consistent with ZmCCAl and ZmTOC1 did not change when environment changes,but the strength of rhythm display gradually diminish.These results indicate that ZmELF4 gene may be a circadian clock gene in maize.3.The plant expression vectors CPB::Z58-ZmELF4-ox and 8W-ZmELF4-ox were constructed and transformed Arabidopsis by Agrobacterium-mediated dip flowers method.The transient expression vectors pC2300::Z58-ZmELF4-GFP and pC2300::18W-ZmELF4-GFP were also constructed and subcellular localization were performed in Tobacco leafs.The results showed ZmELF4 from "Zheng 58" was located in nucleus and ZmELF4 from "18-599 white" was located in cell nucleus and membrane,but predominantly localed on nucleus.