| Mulberry(Morus alba L.)is a perennial woody plant of Moraceae and Morus genus with great economic and ecological importance.It is widely planted all over the world.The floral performance of mulberry trees is complex,most of mulberry varieties are dioecious and diclinism,and some are monoecious and androgynous;in addition,the flower-sex of mulberry are easily affected by the environment,and it is difficult to identify the sex of flower in the early stage,which affects the stability of mulberry fruit production and the genetics breeding of mulberry trees.In the mulberry garden of our school,our research group found that two mulberry varieties,Jialing No.40 and Hongguo No.1,which were dioecious in many years,appeared hermaphroditic buds in 2019,and also showed hermaphroditic buds in the following year.In this study,the male and female flower inflorescence of these two varieties were collected,and the differentially expressed genes of male and female flowers of mulberry were obtained through transcriptome sequencing analysis.The expression characteristics of DEGs at different developmental stages of mulberry floral organs and different tissues and organs of mulberry were analyzed by q RT-PCR expression,which provided preliminary insights into the floral differentiation and development of mulberry at the molecular level.Besides,the excavated Mn ARR17 and Mn PMADS2 genes were further constructed into plant expression vectors,and the biological functions of these two genes were analyzed by heterologous transformation of tobacco.The main findings are as follows:1.The male and female mulberry flower inflorescences of the same buds were sequenced by Illumina high-throughput,and a total of 26.21 Gb of clean reads were obtained.The clean data of each sample is around 6.36 Gb,the percentage of Q30 bases is 94.56% and above,and the GC content is around 46%.Using Trinity software,de novo assembly was performed,and a total of 100,177 Unigenes were obtained by splicing,with a total length to82,311,264 bp,an average length to 821.66 bp,and an N50 to 1,555 bp.Among them,there were 23,246 Unigenes with a length of more than 1kb.Using the BLAST tool to compare Unigenes with public databases,a total of 55,288 Unigenes obtained annotation information from seven major databases.GO and KEGG pathway analysis showed that mulberry flower organ transcripts were mainly expressed in GO items such as cell,binding activity and metabolic process,and were involved in the regulation of 130 metabolic pathways,mainly including carbohydrate metabolism,kinase activity regulation and plant hormone signal transduction.Difference analysis showed that a total of 1,717 DEGs were identified,of which 1,366 were up-regulated in male flowers and 351 were up-regulated in female flowers.Through functional enrichment analysis of DEGs and related literatures had reported,26 genes involved in the regulation of mulberry flowering time were screened,mainly in photoperiod,gibberellin and age pathways;54hormone-related genes that may be involved in the sexual regulation of mulberry flowers,mainly ethylene,cytokinin,gibberellin and auxin for metabolic regulation and signal transduction;there are also a large number of floral organ development genes such as AGAMOUS,PMADS1/2 and AGL11 and etc.And potential sex regulation genes such as ACS,ACO,ARR17 and TASSELSEED2 were annotated,too.Then,we performed SSR and SNP locus polymorphism analysis of all sequences in the mulberry male and female flower sequencing data.There are 30,010 Unigene sequences containing a total of 40,310 SSR sites.All SSRs can be divided into seven types,and the single-nucleotide repeat type has the largest number to 22,850,accounting for 56.69%.And the complete SSR contains 77 types of repeat motifs,mainly A/T mononucleotide repeats,were 27,831,accounting for 62.54%.According to SNP statistics,there are 34,932 Unigene sequences containing a total of 172,126 SNP sites.And 99,943 identical SNP sites in 19,012 Unigene sequences in all samples.Besides,there are more than 20,000 SNP sites that may be involved in floral and cultivar characteristics,respectively.The structural feature types of all SNP sites are divided into two types: transition and transversion,mainly A/G and C/T,each accounting for about 31%,respectively.2.Verify the accuracy of transcriptome data by q RT-PCR and screen some floral regulatory genes to carry out expression profiling analysis.The results showed that the correlation in the real-time fluorescence quantitative data of 20 DEGs with the abundance values characterized by sequencing showed that the transcriptome data was accurate and reliable.The five floral organ development regulatory genes showed obvious functional specificity in the spatiotemporal and tissue expression profiles of mulberry.The up-regulated genes AGAMOUS and AGL11 in female flowers showed a stable up-regulated expression trend with the development of female flowers,and also had higher expression levels in buds;the up-regulated genes PMADS1 and PMADS2 in male flowers with the flowers gradually matured,and their expression levels were higher.After the expression reaching the peak,it decreased rapidly.While AGL104,which is involved in pollen grain development,was mainly expressed at a high level in the pollen maturation stage of the late male flower.The expression characteristics of the rate-limiting enzyme ACS in the ethylene synthesis pathway are highly correlated with the development of mulberry female flowers,while ACO is highly expressed in all tissues;The downstream response gene ARR17 of cytokinin showed its possible different functions in different developmental stages of male and female flowers,and the gene was also significantly enriched in stipules;and the two oxidases in the gibberellin synthesis pathway,the expression trend of GA20Ox1 was consistent with the development of male flowers of mulberry,while the expression trend of GA20Ox3 indicated that this gene may be involved in the development of mulberry female flowers.3.In this experiment,potential woody plant sex-regulating gene ARR17 and type-B floral organregulating gene PMADS2 were successfully cloned.The CDS sequences of the two genes are 465 and606 bp,respectively,encoding 154 and 201 amino acids,respectively.The molecular weights of the two proteins are 23.21 and 17.16 k D,respectively.Then,two overexpression vectors,PLGNL-ARR17 and PLGNL-PMADS2,were constructed and transferred into Nicotiana attenuata for functional verification.GUS staining and molecular identification preliminarily confirmed that the two genes were overexpressed in tobacco lines.Phenotypic observation showed that PLGNL-ARR17 transgenic tobacco plants showed slow growth,abnormal root development,the main root division is enhanced and longer,and the number of lateral roots are reduced.Besides,the pistil stigma of transgenic tobacco is protruding,and very few flower buds with abnormal pistil.While PLGNL-PMADS2 had abnormal stamen development and increased anther number in some flowers,pollen grains were deformed,shrunken or ruptured,and a few flower buds showed abnormal petal development.These results indicate that the Mn ARR17 gene in mulberry is related to the development of floral organs,and whether it has a sex-regulating function needs a further research;while the function of the Mn PMADS2 gene is basically consistent with the the description of floral structure model,and the gene also has a certain degree of functional differentiation. |
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