Cloning And Functional Analysis Of The NAC Gene Of Pinus Massoniana

Currently,researches on NAC gene are mostly focused on stress,and experimental materials are mostly model plants,such as arabidopsis,rice and soybean,etc.,while researches on woody plants are relatively weak,especially conifer species such as Pinus massoniana.Through the full-length transcriptome sequencing of P.massoniana seedling,the NAC gene sequence was obtained,and the expression of NAC gene in the process of drought stress,low phosphorus stress and reproductive growth was analyzed.The role of NAC gene in the stress response process and reproductive growth process of P.massonina was initially explored,which laid a foundation for the diversification study of NAC gene function.The main results are as follows:(1)Whole-length transcriptome sequencing was performed on 1-year seedlings of P.massonina during normal growth and drought stress,a total of 94 NAC genes were obtained,phylogenetic tree construction using published arabidopsis thaliana NAC protein and P.massonina NAC protein,the NAC protein of P.massonina exists in 8 subfamilies,including AtNAC3,NAP,NAM,OsNAC7,NAC2,ONAC003,ANAC011 and PmATAF,among which the PmATAF subfamily is unique to Pinus massoniana.Conserved analysis of the NAC protein of P.massonina showed that the5,6,1,2,and 3 sequences of the 10 NAC proteins of P.massonina were corresponding to the A,B,C,D,and E subdomains of the NAC gene.Most of the members of each subfamily had the same motif,and only part of the conserved motif of P.massonina NAC protein was lost.(2)Through the analysis of the second-generation transcriptome sequencing of P.massonina seedling under drought stress,it was found that 11 of the 94 NAC genes were significantly changed in the expression level under drought stress.Evolutionary tree analysis showed that these 11 NAC genes were clustered into a group,belonging to AtNAC3,PmATAF and NAP subfamily,with high similarity.Combined with bioinformatics analysis,this part of NAC gene was speculated to be related to drought stress.(3)NAC fragments selected from the second-generation transcriptome data(seedlings under low phosphorus stress,microsporidium in sexually reversed)were compared with the full-length transcriptome,and four genes were specifically amplified and verified,which were named PmNAC1,PmNAC2,PmNAC3 and PmNAC4 respectively.One gene was specifically cloned and named as PmNAC5.The total length of PmNAC1-PmNAC5 cDNA was 2717 bp,2257bp,2257 bp,1134bp and822 bp,the open reading frame is 2010 bp,1767 bp,885 bp,1056 bp,822 bp.Codes669 aa,587aa,274 aa,351aa,273 aa.PmNAC1~5 is predicted to be located in the nucleus,which is an unstable protein with hydrophilicity.PmNAC1 and PmNAC2 have transmembrane behavior.(4)Tissue specific expression analysis showed that the expression pattern of PmNAC1 was different under low phosphorus stress than that under very low phosphorus stress.Under low phosphorus stress,the expression pattern of PmNAC1 was increased in needles,decreased in roots and stems,and all tissues were down-regulated under very low phosphorus stress.Under very low phosphorus stress,the expression of PmNAC5 was inhibited in the root,but increased in the old and new leaves.(5)During the transformation of microspore spherules into macrospore spherules,the expression level of PmNAC2 continued to increase,and the expression level of macrospore spherules with complete sexual inversion reached the highest level on multi-spore fruit branches,which was increased by 2.92 times.PmNAC2 and PmNAC4 showed a tendency of high expression of macrosporidium.PmNAC3 was highly expressed in microsporophyll of P.massonina,and was down-regulated to 0.16 times of the original level in megasporophyll.There was a significant gender bias expression,indicating that PmNAC2,PmNAC3 and PmNAC4 were related to gender differentiation and development.In conclusion,NAC gene is abundant in P.massonina and plays an important role in response to drought stress,low phosphorus stress and sex differentiation of sporophyll globules.