Functional Identification Of Flowering-stage Related Gene GhMYB44 From Gossypium Hirsutum

Early maturity is not only an excellent agronomic trait,but also a good quality trait.Therefore,it is of great significance for the development of crop production to discuss how to combine conventional breeding methods and molecular biology techniques to shorten the growth period of major crop varieties.Short growth period is the main character of early maturit cotton,which is insensitive to changes of light and temperature.Therefore,early maturity cotton is suitable for late sowing in spring,even in early summer.The light and heat are abundant with a short period in the inland areas of China.It is a good way to plant early maturity cotton in this area to fully utilize the light and heat resources and improve benefits.Growing early maturity cotton with appropriate cultivation technique will achieve tow or more harvests of cotton fibers and grains a year,increasing land multiple cropping index and use efficiency to alleviate the shortage of cultivated land.Further,it’s helpful to grow early maturity cotton to avoid cold,drought,disease and pest in early spring and autumn,availing the reduce of pesticide and field management to save the cost and labor.However,the germplasm of early maturity cotton is narrow in China,which is a limitation of the development of breeding of early maturity cotton.Thus,investigation of genes regulating flowering time and clarification of their functional mechanism will shed importance in cotton breeding and cotton industry development.In the previous study of our research group,some flowering-stage related genes were obtained.In this study,a MYB gene was cloned,and its function was further investigated.The main results were as follows.1.A full-length of 1322bp fragment was cloned from cotton TM-1,which contained an open reading frame of 987 bp encoding a 37.1 kD protein with 8.93 theoretical isoelectric point.The cloned fragment contains two conserved MYB domain and classified as a R2R3-MYB transcription factor,thus was named as GhMYB44.Phylogenetic analysis identified a homologous with the sequence similarity of 95%thar is located on the A02 chromosome.2.GhMYB44 was expressed in root,stem,leave,and pistils with the highest expression in leaves.The expression of GhMYB44 accumulated till floral transition and displayed higher expression in the SAM.3.The fusion vector 35s:GhMYB44-GFP was constructed and transiently expressed in tobacco leaves.The GFP-GhMYB44 signals were observed in the nuclear,suggesting that GhMYB44 functions in the nuclear,which is consistent with its function as a transcription factor.4.The overexpression vectors 35s:GhMYB44 and 35s:GhMYB44-6HA were constructed and transformed into Arabidopsis thaliana,and finally homozygous were obtained for further investigation.Both Real-time PCR and Western Blot results showed that the target protein has been expressed successfully in Arabidopsis.The transgenic lines all flowered earlier than the wild type under 23℃ long day condition,suggesting that GhMYB44 is a flowering-stage promoter.The VIGS interference vector was constructed and infected with cotton,and 13 plants with a silencing efficiency of 50%were detected by qRT-PCR.Phenotype identification revealed that the time of the first fruit branch node of the silent plant was delayed after silencing the GhMYB44 gene.Then GhMYB44 expression was compared between early and late flowering cottons.Constantly,the mRNA levels were higher in the early-flowering cottons.5.To study the function of GhMYB44 in the regulatory network of flowering time,the response of 35S:GhMYB44 to different ambient temperature,day-length and GA were analyzed.The results showed that 35S:GhMYB44 demonstrated the same early flowering phenotypes under short day,long day condition and GA treatment.But the early flowering phenotype abolished under 27℃ long day.Real-time results showed a significantly decrease of PIF4 and FVE transcripts.Therefore,we speculate that the GhMYB44 gene is involved in the ambient temperature pathway and regulates flowering-stage through PIF4 or FVE.In this study,GhMYB44 was cloned which encodes a R2R3 MYB transcription factor and functions in the nuclear to promote flowering.In normal temperature,GhMYB44 expression increased gradually to promote floral transition,while high temperature could disturb its function,resulting in a decrease of PIF4 and FVE,and thus reduce the expression of flowering time integrator FT to inhibit early-flowering in response to high temperature.