Expression And Functional Characterization Of Genes During Pollen Development And Fertilization In Brassica Campestris Ssp. Chinensis

Pollen development and fertilization are important processes in plant life cycle which directly related to the yield, and seed number and quantity, etc. However, the molecular mechanisms of pollen development and fertilization are quite complex, which involving vast of genes and proteins. Nowadays, the understanding of these two processes is far from enough. Analysis the transcriptome, microRNAs (miRNAs) and gene functions during pollen development and fertilization would shed light on the understanding of these two independent but closely connected processes, and possess essential theoretical and applicable meanings.Three different male sterile lines including ’Aijiaohuang’ genie male sterility AB line (ajhGMS ’Bcajh97-01A/B’),’Polima’ genic-cytoplasmic male sterility (polG-CMS ’Bcpol97-05A’) and ’Ogura’ cytoplasmic male sterility (oguCMS ’Bcogu97-06A’) in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis were used in the present study. Firstly, we analyzed the time courses of four different stages and of fertilization process in ’Bcajh97-01A/B’ plants. Then we identified the differential expressed genes during fertilization in pistils of ’Bcajh97-01A’ using GeneChip(?) Arabidopsis ATH1genome array (ATH1). Then, we analyzed the miRNA expression differences between the3different male sterile lines with their common maintain line respectively, and compared the differential expressed miRNA among the three materials. Meanwhile, the differential expressed miRNAs expressed before and after pollination in pistils were also analyzed. In addition, two Brassica campestris pectate lyase like9(BcPLL9)、BcPLL10and one male novle gene Brassica campestris male fertile21(BcMF21) were selected for further research. The structure, expression pattern, and evolution pattern of the three genes were analyzed. And antisense RNA technology was used to knock down the genes respectively. The morphological, molecular biological and cytological aspects of the antisense RNA transgenic plants were characterized to understand the functional mechanism of the genes. The main results are summarized as following:(1) The time courses of the developmental stages of fertilization in B. campestris ssp. chinensis were identified as four stages. Basing on the microscopy observation, developmental stages of fertilization in B. campestris ssp. chinensis were defined manually as follows:Stage Ⅰ (0～2HAP), pollen grain adhered, hydrated, penetrated into the papillary cells and reached the mid stylar region; Stage Ⅱ (2～4HAP), pollen tubes passed through style and reached at top region of ovary; Stage Ⅲ (4～16HAP), pollen tube reached at bottom region of ovary; Stage Ⅳ (16～24HAP), early embryogenesis.(2) Seventy-seven differential expressed genes were identified during fertilization process in B. campestris ssp. chinensis using ATH1microarray analysis, and ten continuously expressed homologous genes were obtained by comparing the differential expressed genes with pollen development transcriptomes. Forty-four up-regulated genes and thirty-three down-regulated genes were identified in pollinated pistils compared with unpollinated pistils. According to the functional categories,20%of the up-regulated genes might be involved in cell wall development. Ten genes up-regulated in both pollen development and fertilization, such as At2g02720, were selected and termed as continuously expressed genes. Seven of the ten genes were potentially involved in cell wall development, and two of them encoded PLLs (At2g02720, PLL9and At3g01270,PLL10). (3) The miRNAs related to pollen development were identified in inflorescences of three different male sterile lines (ajhGMS ’Bcajh97-01A’,polG-CMS ’Bcpol97-05A’ and oguCMS ’Bcogu97-06A’ compared with their common maintain line ’Bcajh97-01B’) using microRNA μParafloTM icrofludic chip. The differential expressed miRNAs among the three male sterile lines showed partical overlaps. There were11miRNAs which belonged to7families were identified between ’Bcajh97-01A’ and;Bcajh97-01B’ inflorescences (3families down-regulated, and5families up-regulated);64miRNAs which belonged to23families between ’Bcpol97-05A’ and ’Bcajh97-01B’(8families down-regulated, and14families up-regulated);70miRNAs which belonged to20families between ’Bcogu97-06A’ and ’Bcajh97-01B’(10families down-regulated, and11families up-regulated). miR894were down-regulated in all three male sterile lines, and miR172and miR399showed up-regulated in all the three lines. One down-regulated miRNA and four down-regulated miRNAs were detected in both ’Bcajh97-01A ’and ’Bcpol97-05A’; one down-regulated miRNA and two down-regulated miRNAs were detected in both ’Bcajh97-01A’ and ’Bcogu97-06A’; eight down-regulated miRNA and seven down-regulated miRNAs were detected in both ’Bcpol97-05A’ and ’Bcogu97-06A’. This indicated that the three male sterile lines showed different miRNAs regulatory patterns caused by their different genes controlling their male sterile phenotypes. The shared the same nuclear genome background, thus gave several common differential expressed miRNAs. Eight miRNA families were identified as differential expressed after pollination in pistils in B. campestris ssp. chinensis, with six belonged to four families down-regulated and16belonged to four families up-regulated. Moreover, fertilization differential expressed miRNAs were compared with that of pollen development related miRNAs, six miRNA families (miR896, miR1450, miR172, miRNA168, miR396and miR158) were also detected as differential expressed in the inflorescences of the male sterile lines as well in pollinated pistils. In total,30non-redundent miRNAs were identified as differential expressed during pollen development or fertilization, with70%of which have been reported to be expressed in pollen.(4) A pollen and pistil specific expressed gene, BcPLL9, was related with intine development. Full length cDNA and DNA of BcPLL9were obtained by using homologous cloning. Structure analysis indicated that BcPLL9posses the chacater of pectate lyase. qRT-PCR and in situ hybridization showed that BcPLL9expressed in mature pollen grain, stigma and upper style. The pollen was partial abnormal when the expression level of BcPLL9was knocked down by antisense RNA technolpgy. In vitro pollen germination showed that the pollen tube wall could not appressed to pollen tube membrane but in a loosen statue (44.8%), and pollen tubes were shorter in length. In vivo pollen germination showed that pollen tube could grow in style but the ratio it reached the bottom of ovaries was lower which led to lower seed production. Abnormal callose accumulation was observed in BcPLL9antisense RNA transgenic plants. The endintine and exintine structure of intine was not observed in BcPLL9antisense RNA transgenic plants.(5) A pollen and pistil specific expressed gene, BcPLL10, was related to pollen wall development. Full length cDNA and DNA of BcPLL10were obtained by using homologous cloning. Structure analysis indicated that BcPLL10posses the chacater of pectate lyase. qRT-PCR and in situ hybridization showed that BcPLL10expressed in pollen grain, stigma, style and ovary. The tryphine was accumulated out of the tectum and randomly and in the anther locule when the expression level of BcPLL10was knocked down by antisense RNA technology. In addition, the size of the pollen and the localization of germinal furrows were not the same in bcpll10with the empty vector transgenic plants. In germinal furrow, the exintine over developed while the endintine was thinner, Outside of germinal furrow, the layers of exintine and endintine were not clear.(6) PLL genes located on the three subgenomes of B. campetris in different densities. The expression patterns of duplicated BcPLL genes are not exactly the same but relative conserved. BcPLL8-BcPLL11 might be origated from neufunction and subfunction. Eighteen of46BcPLL were located on less fractioned subgenome,16on more fractioned subgenome1,12on more fractioned2, which supporting the the hypothesis that Brassica evolution through two time polyploidation and two time diploidization. PLL8, PLL9, PLL10and PLL11contained a novel N termited conversed region which other PLLs did not. qRT-PCR analysis indicated that PLL8, PLL9, PLL10and PLL11expressed specifically in anther and pistil. It is speculated that they might be origated from gene neufunction and subfunction.(7) Microspore and tapetum specific gene BcMF21played a role during pollen development and pollen tube growth. The gene full length of a167bp cDNA-AFLP screened fragment (A18T19-4, BBP10/BPO054) was isolated with homologous cloning and RACE-PCR. Bioinformatic analysis showed it showed low similarity with other gens, thus reprented as a novel early pollen development related gene, thus named as Brassica campestris male fertile21(BcMF21). Structural analysis showed it might be important for signal transduction. RT-PCR and in situ hybridization results showed that it expressed specifically in microspores and tapetum during meiotic stage, tetrad stage and uninuleate stage. There was48.2%of pollen grains in BcMF21antisense RNA transgenic plants were small sized and shrunken. The left pollen grains were normal sized. However, their surface was not reticulated as the pollen grains in empty vector transgenic plants, but showed a layer of lipid like material nearby the germinal apertures. It is speculated that BcMF21might be functional in pollen development through signal transduction in microspores and tapetal cells.