Gene Mining Of Male Gametophyte Development In Betula Platyphylla Suk.and Genetic Transformation Of BpAMS And Bp034892.1

Betula platyphylla Suk.is one of the important forest trees in Northeast China,which has unisexual flowers,hermaphrodites,and the same-aged androgynous inflorescences do not meet in the flowering period,which is of great research value.The natural birch male inflorescences mutant is the research material of this experiment.The mutant has two male inflorescences,one is born on the top of the branch in line with the wild type,named normal-like male(NLM),and the other is born on the middle part of the branch is named mutant male(MM),and both male inflorescences are aborted.The two male inflorescences of the mutant and the wild-type male inflorescences normal male(NM)were cytologically observed.Their development processes were divided into six stages.The results showed that at the fifth stage of the wintering period,the tapetum cells of the wild-type male inflorescences had begun programmed cell death,and the disintegration of the tapetum cells of the two male inflorescences in mutant was delayed.Finally,in the pollination stage,wild-type male inflorescences produce mature pollen grains,and in the mutant male inflorescences,NLM has some microspore formation,and MM does not produce microspores.Finally,both mutant male inflorescences cannot form viable pollen.In order to investigate the cause of pollen abortion in mutants,we performed transcriptome sequencing on wild-type male inflorescences(NM)and mutant male inflorescences(MM).Based on GO annotation and log2(MM/NM)of transcriptome data,10 genes related to male gametophyte development and differentially expressed in mutants were screened out.Using qRT-PCR,we explored the expression of 10 genes at different stages of male inflorescences development and different tissues.Among them,MYB32,MYB38,AMS,MS1,LTP14,FIL1 and ABCG26 were highly expressed in the fourth stage of mutant inflorescence(MMs)development.Based on the delay in the process of programmed cell death of the tapetum cells in NLM and MM at stage 5,it is speculated that it may be caused by the above genes.MYB32,MYB38,AMS,MS1,LTP14,FIL1,ODO1 and ABCG26 are specifically expressed in male inflorescences.It is speculated that these eight genes play an important role in the development of male gametophyte.The gene BpAMS,which was significantly up-regulated at the fourth stage(the tetrad and microspore stage)of the two inflorescences in mutant and specifically expressed in the male inflorescences,and the Bp034892.1 gene,which was differentially expressed in the transcriptome but unknown in function,was used as candidate genes.The ORF sequences of the BpAMS and Bp034892.1 genes were cloned respectively,and the overexpression vector(35S::BpAMS-GFP,35S::Bp034892.1-GFP)and the suppression expression vector(35S::BpAMS-SRDX,35S::Bp034892.1-SRDX)were constructed.Subcellular localization showed that BpAMS and Bp034892.1 were localized in the cytoplasm and nucleus.Using leaf disc method,35S::BpAMS-SRDX and 35S::BpAMS-GFP were obtained 9 and 4 transgenic lines,respectively.235S::Bp034892.1-GF1P and 35S::Bp034892.1-SRDX were obtained 8 and 3 transgenic lines,respectively.Statistic plant height,leaf length to width ratio and internode distance,found no significant difference between 35S::BpAMS-SRDX and wild type in B.platyphylla.Combined with the expression of BpAMS in wild-type male inflorescences is significantly higher than root,stem,leaf.Suggesting that BpAMS is related to the development of male gametophyte,and has no obvious effect on the early stage of vegetative growth.