Stigma-Specific Comparative Proteomic Analysis Reveals the Distyly Response to Self-Incompatibility in Plumbago auriculata Lam

In plants,heteromorphic self-incompatibility(HetSI)is a strategy for avoiding self-pollination and promoting outcrossing,and during this process,numerous protein-protein interaction events occur between the pistil and pollen.Previous studies in Primula and Fagopyrum that focused on HetSI systems have provided interesting insights;however,the molecular mechanism underlying HetSI remains largely unknown.In this study,we profiled the proteome of Plumbago auriculata stigmas before and after self-incompatible(SI)and self-compatible(SC)pollination.Comparative analyses were conducted by 4D-DIA(Four-dimensional data independent acquisition),a promising technology that increases the sensitivity and reduces the spectral complexity of proteomic analysis by adding a fourth dimension,ion mobility.The results revealed 33387 peptides and 5311 proteins in all samples.The pathways in which the differentially expressed proteins(DEPs)identified in the P×P(Pin style self-pollinated with pin pollen)vs.PS(Pin style)and T×T(Thrum style self-pollinated with thrum pollen)vs.TS(Thrum style)comparisons were significantly enriched were biosynthesis of secondary metabolites and pentose and glucuronate interconversions.In the P×T(Pin style cross-pollinated with thrum pollen)vs.PS and T×P(Thrum style cross-pollinated with pin pollen)vs.TS comparison,the top three pathways were biosynthesis of secondary metabolites,pentose and glucuronate interconversions,and phenylpropanoid biosynthesis.The phenylpropanoid biosynthesis,cutin,suberine and wax biosynthesis,and flavonoid biosynthesis pathways were enriched in the P×T vs.P×P comparison,and starch and sucrose metabolism,glycerophospholipid metabolism,and alpha-linolenic acid metabolism were abundant in the T×T vs.T×P comparison.The enriched pathways between PS and TS were the biosynthesis of secondary metabolites,phenylpropanoid biosynthesis,and pentose and glucuronate interconversion.Self-incompatibility protein S1(SI S1),Mitogen-activated protein kinase 3/4(MPK3/4),Mitogen-activated protein kinase kinase 2/3(M2K2/3),Exocyst complex component EXO70A1(E70A1)and Thioredoxin H1/2(TRXH1/2)were found to be HetSI-related candidates,and O-fucosyltransferase 23(OFT23),3-ketoacyl-CoA synthase 6(KCS6),Receptor-like protein kinase FERONIA(FERON),Fimbrin-5(FIMB5),Pollen-specific leucine-rich repeat extensin-like protein 4(PLRX4),Transcription initiation factor IIB-2(TF2B2)and Pectinesterase 1(AL11A),etc.,were identified as other regulatory transducers.These findings combined with our morphological and reactive oxygen species(ROS)intensity analyses indicate that P.auriculata has typical dry-stigmas and that the HetSI mechanism might differ between the pin and thrum.SI S1 might be the key factor in HetSI,and ROS are overexpressed during SC pollination to rapidly activate the mitogen-activated protein kinase(MAPK)-mediated phosphorylation of E70A1 to maintain stigma receptivity in plants with HetSI.

The Self-incompatibility (S) Locus of Antirrhinum Resides in a Pericentromeric Region

The self-incompatibility ( S) loci from the Solanaceae, Rosaceae and Scrophulariaceae encode a class of ribonucleases, known as S RNases, which have been shown to control the pistil expression of self-incompatible reaction. In the former two families, the S loci have been shown to be located near centromere. However, the chromosomal location of the S locus in Antirrhinum, a species of the Scrophulariaceae, is not known. To determine its chromosomal location and genomic organization, an S-2 RNase gene and its corresponding 63 kb BAC clone were separately used for fluorescence in situ hybridization (FISH) of mitotic metaphase chromosomes of a self-incompatible Antirrhinum line Of S2S5. The results showed that the S-2 RNase detected a doublet signal near the centromere of the smallest chromosome (2n = 16). Two separate doublet signals of the tested BAC sequence were shown on both sides of the centromeres of all eight pairs of the chromosomes, suggesting that the Antirrhinum S locus is located in a pericentromeric region. Furthermore, a retrotransposon, named RIS1 (retrotransposon in the S locus), which has not been identified yet in. Antirrhinum, was found next to S-2 RNase. Taken together, the centromeric location of the S locus from the three S-RNase-based self-incompatible families provides a further support on a common origin of their evolution as well as suppressed recombination.

Recent advances in identification of male specificity determinant and its function in S-RNase-mediated gametophytic self-incompatibility

S-RNase-mediated gametophytic self-incompatibility （GSI） is controlled by a multiallelic S-locus at which two separate genes, the female （pistil） and male （pollen） specificity determinants, are tightly linked. This review described both the identification of pollen specific F-box genes, SLF/SFBs, in Antirrhinum, Petunia and Prunus species and the demonstration of SLF/SFB as pollen determinant together with their functions in GSI response. Recent studies of how the pollen determinant functions in pollination reaction revealed that pollen determinant interacted with S-RNases in a non-allele-specific manner. It targeted all of the non-self S-RNases for ubiquitination through a functional SCF complex and subsequent degradation via 26S proteasome pathway in compatible reaction. It allows pollen tube to reach into the embryo sac and to finish double fertilization. In incompatible response, the intact self S-RNases were left to function as a cytotoxin that degrades self-pollen tube RNA, resulting in the cessation of pollen tube growth.

Identification of Self-Incompatibility Genotypes in Some Sand Pears (Pyrus pyrifolia Nakai) by PCR-RFLP Analysis

The identification of self-incompatibility genotype （S-genotype） will be useful for selection of pollinizers and design of crossing in cultivar improvement of sand pear. This paper reported the identification of self-incompatibility genotypes of seven Chinese and two Japanese sand pear cultivars using PCR-RFLP analysis and S-RNase sequencing. The Sgenotypes of these cultivars were determined as follows： Huali 1 S1S3, Shounan S1S3, Xizilti S1S4, Qingxiang S3S7, Sanhua S2S7, Huangmi （Imamuranatsu） S1S6, Huali 2 S3S4, Baozhuli S7S33, Cangxixueli S5S15. S-RNase alleles （S1 to S9） in sand pear could be identified effectively by PCR-RFLP analysis.

Heterosis of Double Low Self-incompatibility in Oilseed Rape(Brassica napus L.)

66 F 1 hybrids, produced by 3 double low self-incompatible lines and 22 varieties with a North Carolina II (NCII) crossing design, were tested for their heterosis in Wuhan, China during two growing seasons from 1999 - 2001. The results showed that significant differences were found between F1s and their parents for yield per plant and seed oil content. Mid-parent heterosis of these two characters ranged from 5.50% -64.11% and from 1.55% -7.44% respectively. Heterosis for seed yield per plant was greater than that of seed oil content. For yield components, heterosis of total number of siliques per plant was the highest, followed by seed number per silique and 1 000 seeds weight. Significant genotype-by-year interaction was found for seed yield per plant. Results from correlation and combining ability analysis indicated that parental effects on its F! hybrid depended on characters, seed yield per plant was affected by both additive and non-additive effects, and seed oil content was affected mainly by additive effect. When designing hybrid programme, parents might be selected by GCAs and variances of SCAs of parents for the characters affected by both additive and non-additive effects, and by the sum of GCAs of female and male parents for the characters mainly affected by additive effects.

Self-incompatibility Characteristics of 34 Apricot Cultivars in Xinjiang

[ Objective] This study aimed to explore the relationship between self-incompatibility strength and characteristics related to pollination and fertilization of different apricot varieties in Xinjiang. [ Method] The pollen amount, pollen germination rate, pollen tube growth status and fruiting setting rate by self-pollina- tion of 34 apricot cultivars in Xinjiang were determined, to analyze the self-incompatibility of different apricot cultivars. [ Result] The average pollen amount per anther of 34 apricot eultivars was 1 213.7, and the average pollen germination rate was 46.0%. There were great differences in the self-incompatlbility of different cuhivars ; most pollen tubes of the euhivars with high self-incompatibility stopped elongating at 1/3 or 1/2 part of the styles, and only a few pollen tubes of the euhivars with low self-incompatibility reached the ovary, and the normal fertilization ratio was significantly lower than that in self-compatible cultivars. [ Conclusion] Among the 34 apricot cuhivars, only 6 cuhivars were self-compatible and the others exhibited gametophyte self-incompatibility. In addition, the fruit setting rate by self-pollination was low.

Molecular insights into self-incompatibility systems:From evolution to breeding

Plants have evolved diverse self-incompatibility(SI)systems for outcrossing.Since Darwin’s time,consid-erable progress has been made toward elucidating this unrivaled reproductive innovation.Recent advances in interdisciplinary studies and applications of biotechnology have given rise to major break-throughs in understanding the molecular pathways that lead to SI,particularly the strikingly different SI mechanisms that operate in Solanaceae,Papaveraceae,Brassicaceae,and Primulaceae.These best-un-derstood SI systems,together with discoveries in other"nonmodel"SI taxa such as Poaceae,suggest a complex evolutionary trajectory of SI,with multiple independent origins and frequent and irreversible losses.Extensive exploration of self-/nonself-discrimination signaling cascades has revealed a compre-hensive catalog of male and female identity genes and modifier factors that control SI.Thesefindings also enable the characterization,validation,and manipulation of SI-related factors for crop improvement,helping to address the challenges associated with development of inbred lines.Here,we review current knowledge about the evolution of SI systems,summarize key achievements in the molecular basis of pol-len‒pistil interactions,discuss potential prospects for breeding of SI crops,and raise several unresolved questions that require further investigation.

Phase separation of S-RNase promotes self-incompatibility in Petunia hybrida

Self-incompatibility(SI)is an intraspecific reproductive barrier widely present in angiosperms.The SI system with the broadest occurrence in angiosperms is based on an S-RNase linked to a cluster of multiple S-locus F-box(SLF)genes found in the Solanaceae,Plantaginaceae,Rosaceae,and Rutaceae.Recent studies reveal that non-self S-RNase is degraded by the Skip Cullin F-box(SCF)SLF-mediated ubiquitin–proteasome system in a collaborative manner in Petunia,but how self-RNase functions largely remains mysterious.Here,we show that S-RNases form S-RNase condensates(SRCs)in the self-pollen tube cytoplasm through phase separation and the disruption of SRC formation breaks SI in self-incompatible Petunia hybrida.We further find that the pistil SI factors of a small asparagine-rich protein HT-B and thioredoxin h together with a reduced state of the pollen tube all promote the expansion of SRCs,which then sequester several actin-binding proteins,including the actin polymerization factor PhABRACL,the actin polymerization activity of which is reduced by S-RNase in vitro.Meanwhile,we find that S-RNase variants lacking condensation ability fail to recruit PhABRACL and are unable to induce actin foci formation required for pollen tube growth inhibition.Taken together,our results demonstrate that phase separation of S-RNase promotes SI response in P.hybrida,revealing a new mode of S-RNase action.

High-quality Fagopyrum esculentum genome provides insights into the flavonoid accumulation among different tissues and self-incompatibility

Common buckwheat(Fagopyrum esculentum)and Tartary buckwheat(Fagopyrum tataricum),the two most widely cultivated buckwheat species,differ greatly in flavonoid content and reproductive mode.Here,we report the first high-quality and chromosome-level genome assembly of common buckwheat with 1.2 Gb.Comparative genomic analysis revealed that common buckwheat underwent a burst of long terminal repeat retrotransposons insertion accompanied by numerous large chromosome rearrangements after divergence from Tartary buckwheat.Moreover,multiple gene families involved in stress tolerance and flavonoid biosynthesis such as multidrug and toxic compound extrusion(MATE)and chalcone synthase(CHS)underwent significant expansion in buckwheat,especially in common buckwheat.Integrated multi-omics analysis identified high expression of catechin biosynthesis-related genes in flower and seed in common buckwheat and high expression of rutin biosynthesis-related genes in seed in Tartary buckwheat as being important for the differences in flavonoid type and content between these buckwheat species.We also identified a candidate key rutindegrading enzyme gene(Ft8.2377)that was highly expressed in Tartary buckwheat seed.In addition,we identified a haplotype-resolved candidate locus containing many genes reportedly associated with the development of flower and pollen,which was potentially related to self-incompatibility in common buckwheat.Our study provides important resources facilitating future functional genomics-related research of flavonoid biosynthesis and selfincompatibility in buckwheat.

Insights into pollen-stigma recognition:self-incompatibility mechanisms serve as interspecies barriers in Brassicaceae?

A new study provides a comprehensive molecular mechanism that controls interspecific incompatibility of self-incompatible(SI)plants in the Brassicaceae.This finding points to a potentially promising path to break interspecific barriers and achieve introgression of desirable traits into crops from distant species among SI crops in the Brassicaceae.

Molecular Characterization and Expression Analysis of S1 self-incompatibility Locus-linked Pollen 3.15 Gene in Citrus reticulata

Gametophytic self-incompatibility （GSI） is controlled by a highly polymorphic locus called the S-locus, which is an important factor that can result in seedless fruit in Citrus. The S 1 self-incompatibility locus-linked pollen 3.15 gene （S1-3.15） belongs to a type of S locus gene. The role of S1-3.15 in the SI reaction of Citrus has not yet been reported. In this study, full-length sequences of cDNA and DNA encoding the S1-3.15 gene, referred to as CrS1-3.15 , were isolated from ‘Wuzishatangju’ （Self-incompatibility, SI） and ‘Shatangju’ （Self-compatibility, SC） . The predicted amino acid sequences of CrS1-3.15 between ‘Wuzishatangju’ and ‘Shatangju’ differ by only three amino acids. Compared to ‘Wuzishatangju’, three bases were substituted in the genomic DNA of CrS1-3.15 from ‘Shatangju’. Southern blot results showed that one copy of CrS1-3.15 existed in the genomic DNA of both ‘Wuzishatangju’ and ‘Shatangju’. The expression level of the CrS1-3.15 gene in the ovaries of ‘Shatangju’ was approximately 60-fold higher than that in the ovaries of ‘Wuzishatangju’. When ‘Wuzishatangju’ was cross-pollinated, the expression of CrS1-3.15 was upregulated in the ovaries at 3d, and the highest expression levels were detected in the ovaries at 6d after cross-pollination of ‘Wuzishatangju’ × ‘Shatangju’. To obtain the CrS1-3.15 protein, the full-length cDNA of CrS1-3.15 genes from ‘Wuzishatangju’ and ‘Shatangju’ was successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was inhibited significantly with increasing CrS1-3.15 protein concentrations from SI ‘Wuzishatangju’.

Identification of a Ubiquitin-Binding Structure in the S-Locus F-Box Protein Controlling S-RNase-Based Self-Incompatibility

In flowering plants, self-incompatibility （SI） serves as an important intraspecific reproductive barrier to promote outbreeding. In species from the Solanaceae, Plantaginaceae and Rosaceae, S-RNase and SLF （S-locus F-box） proteins have been shown to control the female and male specificity of SI, respectively. However, little is known about structure features of the SLF protein apart from its conserved F-box domain. Here we show that the SLF C-terminal region possesses a novel ubiquitin-binding domain （UBD） structure conserved among the SLF protein family. By using an ex vivo system of Nicotiana benthamiana, we found that the UBD mediates the SLF protein turnover by the ubiquitin-proteasome pathway. Furthermore, we detected that the SLF protein was directly involved in S-RNase degradation. Taken together, our results provide a novel insight into the SLF structure and highlight a potential role of SLF protein stability and degradation in S-RNase-based self-incompatibility.

Turnover of diacylglycerol kinase 4 by cytoplasmic acidification induces vacuole morphological change and nuclear DNA degradation in the early stage of pear self-incompatibility response

Pear has an S-RNase-based gametophytic selfincompatibility(SI)system.Nuclear DNA degradation is a typical feature of incompatible pollen tube death,and is among the many physiological functions of vacuoles.However,the specific changes that occur in vacuoles,as well as the associated regulatory mechanism in pear SI,are currently unclear.Although research in tobacco has shown that decreased activity of diacylglycerol kinase(DGK)results in the morphological change of pollen tube vacuole,whether DGK regulates the pollen tube vacuole of tree plants and whether it occurs in SI response,is currently unclear.We found that DGK activity is essential for pear pollen tube growth,and DGK4 regulates pollen tube vacuole morphology following its high expression and deposition at the tip and shank edge of the pollen tube of pear.Specifically,incompatible S-RNase may induce cytoplasmic acidification of the pollen tube by inhibiting V-ATPase V0 domain a1 subunit gene expression as early as 30 min after treatment,when the pollen tube is still alive.Cytoplasmic acidification induced by incompatible S-RNase results in reduced DGK4 abundance and deposition,leading to morphological change of the vacuole and fragmentation of nuclear DNA,which indicates that DGK4 is a key factor in pear SI response.

Inhibiting Self-Pollen:Self-Incompatibility in Papaver Involves Integration of Several Signaling Events

Cellular responses rely on signal perception and integration. A nice example of this is self incompatibility （SI）, which is an important mechanism to prevent inbreeding. It prevents self-fertilization by using a highly discriminatory cellular recognition and rejection mechanism. Most Sl systems are genetically specified by the S-locus, which has a pollen and a pistil S-component. A receptor-ligand interaction is used by Papaver rhoeas to control SI. S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to achieve rapid inhibition of tip growth. The incompatible Sl interaction triggers a Ca^2＋-dependent signaling cascade. A number of Sl-specific events are triggered in incompatible pollen, including rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases （SPPases）, Prp26.1; activation of a mitogen activated protein kinase, p56; programmed cell death （PCD） involving a caspase-3-1ike activity. These events contribute to prevent self-fertilizaUon. We are attempting to establish the functional significance of these events, and their possible involvement in integrating a coordinated signaling response. Here we describe the identification of these components shown to be involved in Sl, together with recent progress in identifying links between some of them. These data constitute the first steps in elucidating how SI signaling is integrated.

Establishment of Self-incompatibility Gene cDNA Microarray to Identify S-genotypes of Pyrus pyrifolia

Based on the c DNA sequences from hyper variable(HV) regions of identified 52 S-alleles in Oriental pear cultivars, S-RNase c DNA probes were designed, and a c DNA microarray for S-RNase detections was established. Each microarray contained 240 sites from 55 c DNA probes, including all specific c DNA sequences from the HV regions of the S-alleles. Using the c DNA of pistils of tested pear cultivars as template and Cy3 fluorescently labeling primers by PCR amplification, microarray hybridization detected the S-genotype of each pear cultivar. The genotypes inferred from the c DNA microarray hybridization signals of pear cultivars such as ‘Lijiang Huangsuanli', ‘Xiuyu', ‘Midu Yuli', ‘Baimianli', and ‘Deshengxiang' were similar to the known genotypes of all tested cultivars. The S-RNase c DNA microarrays and the oligonucleotide gene chips were then used to conduct parallel testing of 24 P. pyrifolia cultivars with unknown S-genotypes. In conclusion, the construction of c DNA microarrays has further improved the pear S-RNase detection platform.

Identification of S-RNase genotype and analysis of its origin and evolutionary patterns in Malus plants

Identification of the S genotype of Malus plants will greatly promote the discovery of new genes,the cultivation and production of apple,the breeding of new varieties,and the origin and evolution of self-incompatibility in Malus plants.In this experiment,88 Malus germplasm resources,such as Aihuahong,Xishuhaitang,and Reguanzi,were used as materials.Seven gene-specific primer combinations were used in the genotype identification.PCR amplification using leaf DNA produced a single S-RNase gene fragment in all materials.The results revealed that 70 of the identified materials obtained a complete S-RNase genotype,while only one S-RNase gene was found in 18 of them.Through homology comparison and analysis,13 S-RNase genotypes were obtained:S_(1)S_(2)(Aihuahong,etc.),S_(1)S_(28)(Xixian Haitang,etc.),S_(1)S_(51)(Hebei Pingdinghaitang),S_(1)S_(3)(Xiangyangcun Daguo,etc.),S_(2)S_(3)(Zhaiyehaitang,etc.),S_(3)S_(51)(Xishan 1),S_(3)S_(28)(Huangselihaerde,etc.),S_(2)S_(28)(Honghaitang,etc.),S_(4)S_(28)(Bo 11),S_(7)S_(28)(Jiuquan Shaguo),S_(10)S_e(Dongchengguan 13),S_(10)S_(21)(Dongxiangjiao)and S_(3)S_(51)(Xiongyue Haitang).Simultaneously,the frequency of the S gene in the tested materials was analyzed.The findings revealed that different S genes had varying frequencies in Malus resources,as well as varying frequencies between intraspecific and interspecific.S_(3) had the highest frequency of 68.18%,followed by S_(1)(42.04%).In addition,the phylogenetic tree and origin evolution analysis revealed that the S gene differentiation was completed prior to the formation of various apple species,that cultivated species also evolved new S genes,and that the S_(50) gene is the oldest S allele in Malus plants.The S_(1),S_(29),and S_(33) genes in apple-cultivated species,on the other hand,may have originated in M.sieversii,M.hupehensis,and M.kansuensis,respectively.In addition to M.sieversii,M.kansuensis and M.sikkimensis may have also played a role in the origin and evolution of some Chinese apples.

Identification and evolutionary characterization of SFBB genes in‘Yali’and its spontaneous self-compatible mutant‘Jinzhui’(Pyrus bretschneideri)

Pears carry a gametophytic self-incompatibility(SI)system.In this system,S-RNase is the SI pistil determinant,and S-locus F-box brothers(SFBBs)are candidate pollen determinants.However,compared with apple,fewer SFBB genes were identified from pear,possibly caused by the lack of economic and effective methods.Here,we used transcriptome sequencing on‘Yali’(Pyrus bretschneideri)to obtain sequence fragments of SFBB genes and then used polymerase chain reaction(PCR)to amplify the whole sequence of SFBB genes.Twenty-seven SFBB genes,including22 full-length and five nonfull-length SFBB genes,were identified in‘Yali’(P.bretschneideri).SFBBs linkage analysis by PCR-enzyme-linked immunoassay(ELISA)showed that 12 SFBB genes belong to the S21 locus,and 15 SFBB genes belong to the S34 locus.Phylogenetic analysis showed that SFBB genes from Pyrus were divided into 26 types,more than the original eight types.The intrahaplotypic divergence of SFBBs is high and comparable to the allelic diversity of S-RNase,which is consistent with a nonself-recognition SI system.In addition,the expression level of PbrSFBBs in‘Jinzhui’,the only known haploid pollen of a self-compatible mutant,was mostly approximately two times higher than in‘Yali’,which may be the reason for the self-compatible mutant.

Molecular Biology of Self and Non-self Pollen Recognition in Flowering Plants

Fertilization in flowering plants is completed through several recognitionevents, and the first of which is the recognition of pollen by pistil of female reproductivetissue. Self-incompatibility (SI) is an intraspecific reproductive barrier to prevent selfferitilization and widely distributed in flowering plants. In many species, SI shows simplegenetics and is controlled by a single multi-allelic locus, called the S locus. In gametophyticSI (GSI) exemplified by the Solanaceae, Scrophulariaceae and Rosaceae, a class ofribonucleases, called S RNases, have been shown to mediate the stylar expression of SI butnot the pollen expression of SI. The latter appears to be determined by a gene differentfrom those encoding S RNases, often referred to as pollen S gene. The pollen S gene is thecrucial missing part in understanding the biochemical and molecular mechanisms of self andnon-self pollen recognition in flowering plants. Recent genetic analysis of mutationsaffecting the pollen expression of SI has suggested a possible model of how the pollen S geneinteracts with S RNases to achieve self and non-self pollen recognition. Furthermore, wewill present two approaches, S-locus directed transposon tagging and map-based cloning, forcloning the pollen S in Antirrhinum.

Cloning and Bioinformatics Analysis of PsSFBB Gene in Xinjiang Pear

[ Objective] This study aimed to clone the PsSFBB gene from Xinjiang pear for bioinformatics analysis. [ Method ] PsSFBB gene was cloned from an- thers of Qipan pear by using RT-PCR and RACE technologies for bioinformatics analysis. [Result] A SFBBt-α gene with a full-length of 1 231 bp was cloned and named PsSFBB6-α （Genbank accession number： EU909685）. PsSFBBt-ct gene encodes a protein of 378 amino acids, with an F-box motif composed of about 50 amino acids in the N-temfinal. According to the bioinformatics analysis, the molecular formula of PsSFB6-α protein is C2000 H3034 N517 O558S223, with relative molecu- lar mass of 43 987.5 and isoelectric point of 6.02, and the secondary structure is dominated by or-helices ; theoretically, the half life period is 30 h and the instabil- ity parameter is 55.21, so PsSFBB6-α protein is an instable protein ; in addition, it is predicted that PsSFBB6-α protein is a hydrophilic and non-secreted protein with lyases activity and specifically recognized substrates, which was consistent with the function of F-box protein. [ Conclusion] This study laid the foundation for further research on SFBB proteins and the mechanism of self-incompatibility and provided theoretical basis for breeding of self-compatible cultivars of Xinjiang pear and scientific arrangement of pollination trees in production to increase the yield and quality.

Molecular Cloning,Expression Analysis and Localization of Exo70A1 Related to Self Incompatibility in Non-Heading Chinese Cabbage(Brassica campestris ssp.chinensis)

The exocyst is a conserved protein complex,and required for vesicles tethering,fusion and polarized exocytosis.Exo70A1,the exocyst subunit,is essential for assembly of the exocyst complex.To better understand potential roles of Exo70A1 in non-heading Chinese cabbage（Brassica campestris ssp.chinensis）,we obtained the full-length cDNA of Exo70A1 gene,which consisted of 1 917 bp and encoded a protein of 638 amino acids.BlastX showed BcExo70A1 shared 94.9% identity with Brassica oleracea var.acephala（AEI26267.1）,and clustered into a same group with other homologues in B.oleracea var.acephala and Brassica napus.Subcellular localization analysis showed BcExo70A1 was localized to punctate structures in cytosol of onion epithelial cells.Results showed that BcExo70A1 was widely presented in stamens,young stems,petals,unpollinated pistils,roots and leaves of self compatible and incompatible plants.The transcripts of BcExo70A1 in non- heading Chinese cabbage declined during initial 1.5 h after incompatible pollination,while an opposite trend was presented after compatible pollination.Our study reveals that BcExo70A1 could play essential roles in plant growth and development,and is related to the rejection of self pollen in non-heading Chinese cabbage.