Isolating And Expression Analysis Of Candidate Gene PmWND1 Of Weeping Trait In Prunus Mume

Mei(Prunus mume Sieb.et Zucc.)is a traditional and famous flower with great ornamental and cultural value.Weeping trait,as a widely existing variation of branches patterns,has its unique ornamental value.However,the current research about weeping trait of woody plant are focusing on the hormones' effects on branches development and construction of the linkage map to find genetic location using QTL,while the mechanism remain unclear.Previous studies have shown that NAC transcription factors play a critical role in regulating plant secondary development and have significant impact on branches growth.In addition,previous study of our laboratory for QTL of weeping trait in P.mume showed that Pm024260(the homologous gene of Arabidopsis thaliana SND1,named as Pm WND1)is in the candidate region.The effects of Pm WNDl on weeping traits of P.mume were studied in this research.P.mume NAC transcription factor family were carried out with bioinformatic method at the whole genome level.The F1 hybrid population of 'Liuban' ×'Fentaichuizhi' was chosen as the experimental material.The candidate gene PmWND1 was isolated using RT-PCR method and its temporal and spatial expression were analyzed with qRT-PCR.Semi-thin section and paraffin section were employed to make stained tissue slices and thus investigate the cell morphology in second xylem of annual mature branches of mei.The main conclusions are as follows:1.114 NAC genes were identified in P.mume,which were distributed in the 8 chromosome and scaffolds of P.mume.The phylogenetic tree of P.mume and Arabidopsis thaliana NAC was divided into 17 subclasses.NAC genes subfamily tend to have similar gene structure,conserved domains or biological function.Moreover,gene structures of P.mume NAC transcription factors were diverse,most of the genes have 2 or 3 introns,the average number of amino acids was 200 to 400,molecular weight was between 20kDa and 50kDa.2.PmWND1,which encodes 394 amino acids,has the full length of 1185bp and the molecular weight was calculated as 45.46 kDa with DNAMAN.The protein was predicted to be located in the nucleus.The phylogenetic tree analysis showed that PmWNDl was closely related to PpSNDl of P.persica.In addition,the plant expression vector of PmWND1 was successfully constructed using Bgl? and BstE ? double digestion method.3.The expression of PmWND1 increased drastically firstly and then decreased during the branches growth.Meanwhile,for the distribution on the branch,it increased gradually from the shoot top to the base.During early growth period,the expression level of PmWNDl in the middle and the base of upright branches was significantly higher than that in the wepping branches,but there was no difference in the shoots top.In the rapid growth period,PmWNDl was higher expressed on the adaxial side of bending parts and shoots top of weeping branches than that in upright,while in other parts,the expression was the contrary.In addition,the expression of PmWNDl on the branch abaxial side was significantly higher than that on the adaxial side in upright branches,while the contrary observation was shown in weeping.At the end of growth,the expression of PmWND1 in upright branches was still higher than that in weeping,and abaxis side was higher than adaxial side in all parts of both upright and weeping branches.The expression of genes which were related to second cell wall biosynthesis and regulated by PmWND1,were significantly lower in bending part and the base of weeping than those in corresponding parts of upright branches,indicating that PmWND1 was closely related to the secondary cell wall development of second xylem and the formation of weeping traits.4.The secondary cell wall was significantly thicker in the upright branches than that in weeping.Also,in upright branches,the second cell wall in abaxial side was thicker than that in adaxial side.That was consistent with the expression of Pm WND1,indicating that the expression of PmWND1 was positively correlated to the development of secondary cell wall of branch.In this study,the effects of PmWND1 on the weeping trait of P.mume were studied on the basis of the whole genome analysis of the NAC transcription factor.That could provide a reference for the further study on the weeping trait formation of P.mume and woody plants in general.