Cloning And Identification Of Reference Genes And Sex-Related Genes In Gleditsia Microphylla

Gleditsia microphylla Gordon ex Y.T.Lee is a Chinese native species belonging to Leguminosae family,Gleditsia genus.The natural population is mainly distributed in seven provinces along the Taihang Mountains such as Hebei,Shandong and Henan,and now it is widely cultivated as an economic tree species in Cixian and Shexian counties of Hebei Province.Gleditsia microphylla is a dioecious tree species and the seed endosperm produced by female plants is rich in galactomannan,which is a natural chemical raw material and has important economic value.However,the ratio of female to male plants in natural or artificial forest of Gleditsia microphylla is close to 1:1.34,and the excessive male plants seriously affects the planting benefits.Therefore,it is of great theoretical and practical significance to study the sex differentiation and determination of Gleditsia microphylla and develop techniques or molecular markers to distinguish gender at seedling stage.Firstly,the most stable normalizing genes and their combinations used in q RT-PCR analysis in different tissues were identified based on the RNA-Seq data of Gleditsia microphylla in this study,and then validated with the stress-responding genes;subsequently,the top10 genes with high expression level in male and female flowers according to the RNA-Seq data were cloned respectively;to make sure the reliability of RNA-Seq data for their expression levels,they were analyzed further in flowers by q RT-PCR and their correlations to sex differentiation were detemined.Afterwards,the sex-related molecular markers were developed according to their genomic DNA sequences.Finally,the anther microstructure of male and female flowers of Gleditsia microphylla was observed,and the development stage and reasons of abortion of functional female flowers were analyzed,which would provide cytological evidence for the functional female flowers.The main results of this study are as follows:1.According to the RNA-Seq data of Gleditsia microphylla,twelve candidate reference genes（TBP1,ACT7,CBP20,EF-1α,EIF4A1,GAPA,GAPB,PTB1,TIP41,ACT1,e IF2,TUB1）and three stress-responding genes（ACO1,PYL1,CSD2）were selected and cloned.Subsequently,the expression level of these genes in root,stem and leaf tissues of seedlings treated with heavy metals（Cu SO₄）,salt stress（Na Cl）,hormones（Me JA,SA,ABA）,cold and heat（4℃,42℃）and simulated drought（PEG6000）were analyzed using q RT-PCR,and then the stability as a reference gene was determined with four softwares（ge Norm,Norm Finder,Best Keeper,Ref Finder）.The results showed that TBP1 and EIF4A1 were the two most stable reference genes without regarding to treatments and tissues,while GAPA and GAPB were the two most unstable ones.Meanwhile,the most stable internal reference genes vary greatly in different treatments and tissues,TBP1 is the most stable reference gene in flower tissues.2.Based on the RNA-Seq data,eight genes with high expression in female flowers（F444,F1760,F3203,F5382,F5879,F5936,F6087,F9790）and nine genes in male flowers（M18,M865,M978,M4164,M4658,M11713,M16089,M16199,M28688）were selected and cloned,and then their expression levels were detected by q RT-PCR using TBP1 as the reference gene in male and female flowers.It was found that the expression levels of F1760,F5879,F5936,F2893,F444,F6087,M11713,M18,M865,M4164,M978,M28688,M16199,M4658,and M3821 were consistent with the RNA-Seq data,which implied their corelations to the sex differentiation or determination.3.Using the molecular markers designed based on the SNP,SSR and In Del differences between the genomic DNA sequences of the above differential genes in male and female plants,identification with known gender population was carried out,and the results showed that M18,M865,M978,M4658,M11713,M16199,M28688,M4164,F1760,F2893,F5879,and F5936 were heterozygous in both male and female plants,all of them could not effectively distinguish gender;fortunately,the specific primers designed for F444 can amplify the same small fragment product（1500 bp）in female plants and the same large fragment product（1800 bp）in male plants,showing gender correlation.It is speculated that the involvement of F444 in gender development regulation may occur at the transcription or translation level.4.Comparative cytological observation on anther slice of female and male flowers of Gleditsia microphylla at different developmental stages using semi-thin sections demonstrated that the secondary sporogenous cells disintegrated in female flowers,resulting in the stagnation of male gametophyte development,which may be the main cause of the formation of functional female flowers;while the secondary sporogenous cells of male flowers can develop normally and eventually form fertile pollen grains.