Comparative Transcriptomics Analysis Between Cytoplasmic-Nuclear Male Sterile Line NJCMS1A And Its Maintainer And Functional Study Of Fertility Related Genes In Soybean(Glycine Max(L.)Merr.)

Soybean is an important grain and oil dual use crop in the world.At present,the production of soybean in our country is low and cannot meet the needs of our population,which mainly relies on imports.Heterosis is one of the effective ways to improve crop yield,soybean as a self pollinating plant,the breeding of cytoplasmic-nuclear male sterility（CMS）line can effectively solve the difficulties of artificial male castration.However,compared with rice and other crops,the study of soybean cytoplasmic-nuclear interaction male sterility is still not perfect.To investigate the mechanism of male sterility of soybean cytoplasmic-nuclear interaction,comparative transcriptomic analysis and functional studies of fertility related genes GmHB14 and GmWUS between the CMS line NJCMS1A and its maintainer were proposed.The main results are as follows:1.Comparative transcriptomics analysis between cytoplasmic-nuclear male sterile line NJCMS1A and its maintainerThe library was built for transcriptome sequencing using a deribosomal RNA approach,a total of 39.26 Gb of clean reads were obtained from the CMS line NJCMS1A and the maintainer NJCMS1B soybean,which were aligned to the soybean reference genome（Wm82.a2.v1）,resulting in 56213 genes.Using the two criteria of FDR<0.05 and|log₂FC|>1 to screen the differentially expressed genes,a total of 1625 differentially expressed genes were identified between the CMS line NJCMS1A and its maintainer NJCMS1B,223 upregulated and 1402 downregulated genes were identified in the sterile line NJCMS1A compared with the maintained line NJCMS1B.GO functional analysis showed that 474 differentially expressed genes were enriched to 33 GO functional groups,including catalytic activity,ion binding and metabolic processes.KEGG pathway analysis showed that 334 differentially expressed genes were enriched into 90 KEGG pathways,including pentose and glucuronic acid interconversion,starch and sucrose metabolism,oxidative phosphorylation.Combined with relevant literature reports,we found that male infertility in the infertile line NJCMS1A may be associated with abnormal expression of genes involved in mitochondrial development,pollen wall development,carbohydrate metabolism,and reactive oxygen species metabolism.2.Cloning and functional study of the GmHB14 geneBioinformatics analysis indicated that GmHB14 protein has a HOX domain that regulates plant development and a START domain that regulates lipid binding,GmHB14promoter sequence analysis revealed that it has homeostatic action elements such as HD-Zip.q RT-PCR analysis showed that GmHB14 gene was upregulated in flower buds of the CMS line NJCMS1A relative to the maintainer NJCMS1B.Subcellular localization results showed that GmHB14 protein was localized in the nucleus.Fertility analysis results showed that pollen of GmHB14 overexpression of transgenic Arabidopsis was shriveled,aborted,anthers were not powdered,and corneas were shorter.Under salt stress,GmHB14overexpression of transgenic Arabidopsis prematurely bolting with increased total root length,apical and lateral root numbers;Reactive oxygen species staining revealed that DAB staining of leaves and inflorescences of GmHB14 overexpression of transgenic Arabidopsis became lighter,and NBT staining did not change significantly.GUS staining results showed that GUS protein was expressed in stems,leaves,and anthers of GmHB14Pro::GUS transgenic Arabidopsis.Fertility analysis results found that pollen from GmHB14 overexpression of transgenic soybeans was shriveled,aborted,anthers were not powdered,producing a large number of flesh pods and a few individual pods.Based on the above results,it is speculated that the GmHB14 gene may play an important role in plant male infertility and may play a positive role in the salt stress regulatory network.3.CRISPR/cas9 editing and functional study of the GmWUS geneBioinformatics analysis indicated that the GmWUS protein has a HOX domain that regulates plant development.q RT-PCR analysis showed that GmWUS gene was downregulated in flower buds of the CMS line NJCMS1A relative to the maintainer NJCMS1B.Construction of CRISPR/cas9 editing vector for GmWUS gene genetic transformation of soybean was carried out using Williams 82 as recipient parent,CRISPR/cas9 editing soybean for GmWUS gene was obtained and named p CRI-GmWUS.The results of target sequence analysis showed that the GmWUS gene in p CRI-GmWUS soybeans was successfully edited,among which,1 bp base insertion occurred in target 1,8bp base deletion and a single base substitution occurred in target 2,and 2 bp base insertion and a single base substitution occurred in target 3.Phenotypic observations revealed that p CRI-GmWUS soybean exhibited significantly reduced plant height,whereas pollen fertility and plant pod setting were normal.Based on the above results,we found that the GmWUS gene may be involved in the regulation of soybean strain type.