Functional Analysis Of Tillering-related Gene PvMAX2 In Switchgrass

Switchgrass（Panicum virgatum L.）,belongs to Gramineae Panicum,is a kind of perennial grass and native to North American.Switchgrass has two ecological types including highland and lowland.It was widely planted as a new alternative energy plant and an ornamental pasture,because of its special environmental benefit s such as:increased high yield,strong drought and salinity tolerance,extensive ecological adaptability.Tillering（branching）is one of the main factors that affect crop yield and plant type.The researches on switchgrass tillering has begun in recent years.However,the molecular mechanism of tillering regulation in switchgrass was still unclear.MAX2,an F-box protein,was a key factor in SLs signaling pathway.It was involved in a series of functional regulation of plant development.MAX2 has been researched in many specials such as Arabidopsis,rice and pea.The mutant max2 shows more axillary branches,shorter and more lateral inflorescences.In order to study the molecular mechanism of SLs regulating tillering in switchgrass,we have isolated the Pv MAX2 gene from Alamo switchgrass.The results were as follows.1.With the multiple sequence alignment of MAX2,we found PvMAX2 had 92.60%identities to SiMAX2,which means that PvMAX2 is most closely related to millet SiMAX2.Complementation experiment approach of the Arabidopsis max2 showed that Pv MAX2 and At MAX2 gene were similarity functionally.Pv MAX2 gene can increase plant height,reduce tillering number,increase leaf area,facilitate earlier blooming,reduce the number of rosette leaves,accelerate the leaf senescence,inhibit the length of hypocotyl and growth of lateral root,promote root growth and development.2.Effects of GR24 showed that 10^–6M GR24 could increase the length of WT and Pv MAX2-OE transgenic plants primary root,and GR24 inhibited the lateral root and hypocotyl lengths of WT and transgenic plants.Effects of IAA showed that 10^–7M IAA could inhibit the development of primary root and hypocotyl,but it could increase the number of lateral root.The results showed that GR24 regulated the growth of the primary root and hypocotyl in a dependent of Pv MAX2 gene.IAA might regulate the growth and development of the primary root,hypocotyl and lateral root at the downstream of Pv MAX2 gene.3.The expression patterns of SLs gene showed that the expression level of Pv MAX2/D53/D14 were sensitive to GR24 treatment,and and the expression level reached the highest at the 10M of GR24 concentration.D10/D27 showed similarly expression changes that the expression were reduced when conce ntration was higher than 10^-5M.The results showed that there was SLs biosynthesis and signaling pathway in swithgrass,and Pv MAX2played an important role in SLs signaling pathway.4.To investigate the function of Pv MAX2,the Pv MAX2-OE and Pv MAX2-RNAi construct was introduced into switchgrass.We have obtained 9 transgenic plants including 5RNAi plants and 4 overexpression plants after PCR results.In conclusion,Pv MAX2 has a conserved function in regulating plant shoot branching,root and leaf development.Pv MAX2 gene can complement function of At MAX2 and have similarity functions in different plant species.These results will provide the theory basis for SLs regulating the tillering of perennial graminaceae plant.