Cloning And Identification Of Male Sterile Genes BrGGL7 And BrRBL3 In Chinese Cabbage (Brassica Rapa Ssp. Pekinensis)

Plant male sterility refers to the phenomenon that the pistil function is normal but the stamen cannot form functional pollen in flowering plants.Chinese cabbage(Brassica rapa L.ssp.pekinensis)is a bisexual plant with cross pollination and has significant heterosis.Male sterile line is an ideal material to solve the problem of hybrid seed production in the utilization of heterosis.The pure line ‘FT’ of double haploid(DH)of Chinese cabbage was used as test material,and the seeds of ‘FT’ were mutated by ethyl methanesulfonate(EMS)to create male sterile mutants.In this study,two mutants,ftms1 and ftms2,were selected as test materials.Based on the analysis of their pollen abortion phenotype and identification of genetic characteristics,the sterile genes were analyzed using Mut Map gene mapping,KASP genotyping,and q RT-PCR gene expression.The function of the mutant genes was verified by Arabidopsis complementary verification.The main results are as follows:1.Identification of male sterile mutants in Chinese cabbageThe germinating seeds of DH line ‘FT’ of Chinese cabbage were mutated with 0.8% EMS aqueous solution,and multiple male sterile mutants with stable inheritance were created.Two male sterile mutants,ftms1 and ftms2,were selected for morphological and anatomical identification.In the vegetative growth stage,there was no significant difference between mutants ftms1 and ftms2 and wild-type ‘FT’;In the reproductive growth stage,the anthers of ftms1 and ftms2 degenerated and there was no pollen formation.Genetic analysis showed that the stamen sterility of ftms1 and ftms2 were controlled by single recessive genic gene.Paraffin section found that the tapetum of ftms1 was abnormally expanded and vacuolated,and the programmed cell death of tapetum in ftms2 was delayed,both of tapetum in mutants could not provide nutrients for the normal development of microspores,thus leading to microspore abortion.2.The candidate gene of male sterile mutant ftms1 was identified as Bra A05g022470.3C(Br GGL7)by Mut Map and KASP technologies.Its function was verified by Arabidopsis heterologous complementarity experiment.The candidate gene of male sterile mutant ftms1 in Chinese cabbage was identified as Bra A05g022470.3C(Br GGL7)by Mut Map and KASP analyses.The Arabidopsis homologous gene of Bra A05g022470.3C is GGL7,which encodes GDSL lipase and participates in the lipid metabolism of pollen exine.Bra A05g022470.3C from mutant ftms1 showed a non-synonymous SNP mutation(C-T)at the conserved domain resulting in Q(CAA)changing a termination codon(TAA)and leading to truncated protein production.q RT-PCR results showed that Bra A05g022470.3C had the highest expression level in anther,and its expression in ftms1 is lower than that of ‘FT’.Arabidopsis heterologous transformation experiment indicated that Chinese cabbage wild-type gene Bra A05g022470.3C,which named Br GGL7,could restore the pollen fertility of Arabidopsis male sterile mutant.3.The transcriptome of male sterile mutant ftms1 was sequenced by RNA-seq technology,and 76 specifically expressed genes with unknow function and 12 differentially expressed genes related to anther and pollen development were screened.The anthers of wild-type ‘FT’ and male sterile mutant ftms1 were analyzed by RNA-seq.A total of 6,004 differentially expressed genes were identified between ftms1 and ‘FT’.Among these,776 differential genes were specifically expressed,149 were specifically expressed in ftms1 and 627 were specifically expressed in ‘FT’,there are 76 genes with unknown functions.In addition,12 differentially expressed genes related to anther and pollen development were determined,AMS(Bra A03g043400.3C)related to tapetum development,Cal S5(Bra A09g010050.3C)related to callose development,AGP6(Bra A03g006290.3C),AGP11(Bra A03g030350.3C),and FLA3(Bra A04g017980.3C and Bra A09g053670.3C)related to intine development,PIP5K4(Bra A04g003670.3C,Bra A07g022610.3C,and Bra A09g047600.3C)and PIP5K5(Bra A03g021840.3C and Bra A05g002080.3C)related to pollen tube development,and NST1(Bra A04g032280.3C)related to anther dehiscence,and their expression was significantly down regulated in ftms1 compared with ‘FT’.Eight significantly enriched GO terms related to cell and pollen wall and lipid metabolism,and eight significantly enriched pathways related to carbohydrate metabolism and lipid metabolism,which may be related to pollen development,were identified by enrichment analysis of GO and KEGG.4.The male sterile gene of male sterile mutant ftms2 was identified as Bra A10g029960.3C(Br RBL3)by Mut Map mapping and SNP genotyping,its function was verified by Arabidopsis heterologous complementary transformation experiment.The candidate gene Bra A10g029960.3C of male sterile mutant ftms2 in Chinese cabbage was identified by Mut Map mapping and genotyping of SNP.Arabidopsis homologous gene rbl3 of Bra A10g029960.3C encodes rhomboid-like protein 3.Arabidopsis heterologous complementation experiment showed that Chinese cabbage wild-type gene Bra A10g029960.3C could restore the pollen fertility of pollen abortion mutant in Arabidopsis,which proved that Bra A10g029960.3C was gene that caused ftms2 male sterility,so namely Br RBL3.Compared with the wild-type ‘FT’,a non-synonymous SNP mutation(C-T)was determined in the first exon of Br RBL3,which caused the substitution of amino acids from P to S,resulted in the absence of a benzene ring in the protein conformation of the mutation site in mutant.q RT-PCR analysis showed that Br RBL3 was highly expressed in anther,and its expression level in ftms2 was lower than that of ‘FT’.Br RBL3 was mainly located in the nucleus and had a small amount of fluorescence signal in the cytoplasm.